Mutational spectrum and clinical features in 35 unrelated mainland Chinese patients with GNE myopathy.

JNS-13757; No of Pages 6 Journal of the Neurological Sciences xxx (2015) xxx–xxx

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Mutational spectrum and clinical features in 35 unrelated mainland Chinese patients with GNE myopathy Juan Zhao a,1, Zhaoxia Wang a,1, Daojun Hong b, He Lv a, Wei Zhang a, Juanjuan Chen a, Yun Yuan a,⁎ a b

Department of Neurology, Peking University First Hospital, Beijing, China Department of Neurology, The First Affiliated Hospital of Nanchang University, Jiangxi Province, China

a r t i c l e

i n f o

Article history: Received 12 December 2014 Received in revised form 18 April 2015 Accepted 20 April 2015 Available online xxxx Keywords: GNE myopathy Distal myopathy with rimmed vacuoles GNE gene Common mutation Novel mutation Muscle magnetic resonance imaging

a b s t r a c t GNE myopathy is an autosomal recessive distal myopathy caused by biallelic mutation in the GNE gene. It shows great genetic heterogeneity among different ethnic groups. In this study, we summarized the mutational spectrum and clinical profiles in 35 unrelated GNE myopathy patients from mainland China. Molecular analysis revealed 16 novel (p.G47D, p.F66Y, p.E173A, p.Y186H, p.R246L, p.R263*, p.R306*, p.A366D, p.V512M, p.C520Y, p.G545R, p.G548S, p.V622G, p.A638P, IVS2 + 1G N A and c.2112delC) and 13 reported mutations. Notably, the p.D176V mutation was detected in 65.7% (23/35) of this patient cohort, giving an allele frequency of 34.3% (24/70). We estimated the carrier frequency of p.D176V to be 0.19% (1/520) in the normal population, although haplotype analysis indicated no founder effect in the patients carrying p.D176V mutation. Clinically, 29 patients presented with the classic phenotype of predominant distal weakness, while six patients presented with atypical phenotype. However, muscle magnetic resonance imaging showed that the vastus lateralis was spared in both subgroups. In conclusion, p.D176V mutation in the GNE gene, which was the second most common mutation in Japanese patients, was the most common mutation in this cohort of Chinese patients. Novel GNE mutations found in this study expanded the mutational spectrum associated with GNE myopathy. There is phenotypic heterogeneity among patients with GNE myopathy, but muscle magnetic resonance imaging can be useful for differential diagnosis. © 2015 Published by Elsevier B.V.

1. Introduction GNE myopathy is a rare, recessively inherited, adult-onset distal myopathy, also known as distal myopathy with rimmed vacuoles (DMRV), Nonaka myopathy, or hereditary inclusion body myopathy (HIBM) [1]. The causative gene for the disease is GNE, which encodes UDP-N-acetylglucosamine 2 epimerase/N-acetylmannosamine kinase, a bifunctional enzyme in the sialic acid synthetic pathway [2]. GNE myopathy is typically characterized by initial involvement of the tibialis anterior (TA) muscle while the quadriceps is spared, but the myopathy gradually spreads to other muscles. Serum creatine kinase (CK) is usually within normal limits or mildly elevated. The myopathological features are the presence of muscular dystrophy with rimmed vacuoles in the muscle fibers and an absence of inflammatory cell infiltration [3]. GNE myopathy has been reported worldwide, with the vast majority of cases in those of Iranian–Jewish descent and the Japanese [2,4–9]. Abbreviations: GNE, UDP-N-acetylglucosamine 2 epimerase/N-acetylmannosamine kinase; DMRV, distal myopathy with rimmed vacuoles; HIBM, hereditary inclusion body myopathy; TA, tibialis anterior; CK, creatine kinase; MRI, magnetic resonance imaging. ⁎ Corresponding author at: Department of Neurology, Peking University First Hospital, Beijing 100034, China. Tel.: +86 10 83572110; fax: +86 10 66551107. E-mail address: [email protected] (Y. Yuan). 1 These authors contributed equally to this work.

While most patients present with a typical distal weakness phenotype, some patients have an atypical phenotype, such as limb-girdle weakness or asymmetric hand weakness [10,11]. To date, over 150 mutations have been reported across the whole coding region of the GNE gene [12]. There is great genetic heterogeneity of GNE myopathy among different ethnic descents. The c.2135T N C (p.M712T) is named as Middle Eastern mutation, because it appears predominantly in those of Middle Eastern descent [2], while c.1714G N C (p.V572L) and c.527A N T (p.D176V) are most common in Japanese patients [4]. Until now there have been limited data on cases of GNE myopathy from mainland China. Since our group first reported GNE mutations in two patients with DMRV in 2006 [13], Li and his colleagues have reported that c.1523T N C (p.L508S) had an allele frequency of 25% in a group of six unrelated patients, indicating that the common GNE mutations in GNE myopathy patients in China may be different from those in Japan [14]. Later, Lu et al. reported a series of Chinese DMRV patients and found two other common mutations: p.D176V and c.1892C N T (p.A631V), with their allelic frequency being 14% and 15%, respectively [15]. Clearly, more cases are needed to clarify the characteristics of GNE mutations in people from mainland China. Herein we describe the GNE mutational spectrum in 35 unrelated Chinese patients with GNE myopathy, as well as their clinical features and muscle magnetic resonance imaging (MRI) findings.

http://dx.doi.org/10.1016/j.jns.2015.04.028 0022-510X/© 2015 Published by Elsevier B.V.

Please cite this article as: Zhao J, et al, Mutational spectrum and clinical features in 35 unrelated mainland Chinese patients with GNE myopathy, J Neurol Sci (2015), http://dx.doi.org/10.1016/j.jns.2015.04.028

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Case Sex Age no. at onset (y)

Age at exam (y)

Family history (AR)

Onset symptoms

1 2 3 4 5 6

F F M M F F

22 43 40 35 33 43

26 53 44 37 43 47

– – – – – –

7 8

M M

37 39

39 44

– –

Foot drop Foot drop Foot drop Foot drop Walking unsteadily Difficulty in lifting right arm Foot drop Difficulty in running

9

M

26

28

+

10

F

29

30

11 12 13 14 15 16 17 18 19 20 21 22 23

M F F M M F M F F F F M M

30 43 26 23 20 30 31 24 32 32 27 21 33

36 53 34 31 23 57 32 29 39 39 34 28 38

24 25

M M

20 20

26 27 28 29 30 31 32 33 34

F M M F F F F F M

35

F

Muscle strength (MRC grade)

Serum CK (IU/L)a

GNE mutations Allele 1

Allele 2

Site 1

Site 2

4 3 5− 4− 4 5−

462 517 519 424 472 323

c.737G N T (p.R246L) c.527A N T (p.D176V) c.527A N T (p.D176V) c.527A N T (p.D176V) c.527A N T (p.D176V) c.527A N T (p.D176V)

c.1865T N G (p.V622G) c.1571C N T (p.A524V) c.1571C N T (p.A524V) c.1559G N A (p.C520Y) c.1714G N C (p.V572L) IVS2 + 1G N A

Epimerase Epimerase Epimerase Epimerase Epimerase Epimerase

Kinase Kinase Kinase Kinase Kinase Epimerase

5 L:3-; R: 4 5

438 407

c.527A N T (p.D176V) c.527A N T (p.D176V)

c.527A N T (p.D176V) c.1571C N T (p.A524V)

Epimerase Epimerase Epimerase Kinase

5

1 L:3-;R: 4 3

425

c.22C N T (p.R8*)

Epimerase Kinase

4−

5

3

5

476

Kinase

Kinase

5− 5 4 3 4 4 5 5 4+ 5 5− 5 5

3+ 4 4+ 4 3 3 3 4+ 4− 3+ 3 3 4

5 5 4 4 4 4 3 5 5 5 5 4− 5

0 1 4− 4 3 0 4+ 0 0 0 1 4+ 0

3 4 5− 4 3 4 5 4 1–2 2 3 4− 5

2459 429 NA 1792 298 324 1242 254 392 NA 400–730 338 686

c.1534G N A (p.V512M) c.1525C N T (p.H509Y) c.527A N T (p.D176V) c.527A N T (p.D176V) c.1760T N C (p.I587T) c.529C N T (p.R177C) c.527A N T (p.D176V) c.518A N C (p.E173A) c.527A N T (p.D176V) c.527A N T (p.D176V) c.527A N T (p.D176V) c.527A N T (p.D176V) c.197T N A (p.F66Y) c.527A N T (p.D176V)

c.2086G N A (p.V696M) c.1642G N A (p.G548S) c.1525C N T (p.H509Y) c.1714G N C (p.V572L) c.527A N T (p.D176V) c.1760T N C (p.I587T) c.556T N C (p.Y186H) c.1912G N C (p.A638P) c.1912G N C (p.A638P) c.1571C N T (p.A524V) c.1097C N A (p.A366D) c.787C N T (p.R263*) c.1559G N A (p. C520Y) c.698T N C (p.F233S) c. 2112 del C (p.A705fs)

Kinase Epimerase Epimerase Kinase Epimerase Epimerase Epimerase Epimerase Epimerase Epimerase Epimerase Epimerase Epimerase

Kinase Kinase Epimerase Kinase Epimerase Kinase Kinase Kinase Epimerase Epimerase Kinase Epimerase Kinase

5 5

5 4−

5 4

5 5

4− 3

5 5

366 256

c.1571C N T (p.A524V) c.38G N C (p.C13S)

4 5 4 4− 4− 5 5− 5 5−

4− 5 4− 4− 5− 5 5 5− 4

3 5 4 3 5 5 5− 3 4−

1 5 4− 1 4− 3+ 4− 4− 3−

4 5 5 5 5 5 5 5− 3+

1 L:2;R:4 0 1 4− 3 2 0 0

1 L:4; R:5 0 5− 4− 4+ 4− 3+ 2

176 NA 597 198 526 492 1423 572 636

c.38G N C (p.C13S) c.1571C N T (p.A524V) c.527A N T (p.D176V) c.527A N T (p.D176V) c.80C N T (p.P27L) c.527A N T (p.D176V) c.527A N T (p.D176V) c.140G N A (p.G47D) c.527A N T (p.D176V)

5

5

5

4

5

1

3

327

c.527A N T (p.D176V)

Neck Shoulder Hand Iliopsoas QA flexors

TA

GC

5 4+ 4 5 5 4

4 5 4 5 5 2

5 5− 5 5 5 4−

4 4− 4 5 4 4

5 5 5 5 5 5

4 3 3 2 3− 2

4− 4

5 5

5 5

4 4

4 5

Foot drop

4

5

5

5

Foot drop

5

5

5

+ – – + – + + – – + – – +

Walking unsteadily Foot drop, Foot drop Foot drop Foot drop Frequent wrestling Difficulty in going upstairs Difficulty in going upstairs Weakness of both legs Frequent wrestling Weakness of both legs Difficulty in climbing stairs Foot drop

4 5 4 4 4 2 4 4 5 5 5 5 4

5− 5− 4+ 4 5 4 5 4 5− 5 5 4 5

22 27

– –

5 5−

39 42 28 36 22 39 23 29 25

41 45 33 66 24 42 26 36 47

– – – – – – – – –

Weakness of both legs Weakness of hands and feet Weakness of both legs Foot drop Foot drop Weakness of both legs Foot drop Foot drop Foot drop Foot drop Foot drop

30

33

–

Foot drop

AR, autosomal recessive; CK, creatine kinase; QA, quadriceps; TA, tibialis anterior; GC, gastrocnemius; NA, not available. a Normal value: 70–170 IU/L.

Domain

c.1633G N C (p.G545R) c.38G N C (p.C13S) c.916C N T (p.R306*) c.556T N C (p.Y186H) c.527A N T (p.D176V) c.910G N A (p.G304R) c.1523T N C (p.L508S) c.527A N T (p.D176V) c.1534G N A (p.V512M) c.1642G N A (p.G548S)

Kinase ? Epimerase Kinase Epimerase Kinase Epimerase Epimerase Epimerase Epimerase Epimerase Epimerase Epimerase

Epimerase ? Epimerase Epimerase Epimerase Epimerase Kinase Epimerase Kinase

Epimerase Kinase

J. Zhao et al. / Journal of the Neurological Sciences xxx (2015) xxx–xxx


Table 1 Clinical features and GNE mutations (NM_005476) in 35 unrelated Mainland Chinese patients with GNE myopathy.


2. Subjects and methods 2.1. Subjects During 2001–2014, we enrolled 35 unrelated patients diagnosed with GNE myopathy (Table 1). Patients 1–31 were from Peking University First Hospital and patients 32–35 from Nanchang University First Hospital. Among them, we have previously reported on two (Patients 5 and 11) [13]. All 35 patients were of Chinese Han origin, from 15 different provinces of mainland China. All patients had at least five of the following: 1) sporadic or autosomal recessive inheritance; 2) onset in the second to fourth decade of life; 3) preferential weakness of the TA muscle with relatively selective sparing of the quadriceps in early-stage disease; 4) mild to moderate elevation of serum CK; 5) muscle biopsy showing a pattern of muscular dystrophy with the presence of rimmed vacuoles in the muscle fibers; and 6) homozygous or compound heterozygous mutations in the GNE gene. All patients underwent detailed clinical evaluation, including family history, onset age and symptoms, distribution of muscle weakness and serum CK. In patients presenting with non-classical GNE myopathy, mutations of DES, VCP and MYOT had previously been excluded. To evaluate the patterns of muscle involvement, 13 patients underwent MRI of the leg muscles. This study was undertaken with institutional ethical approval. GNE gene mutation screening and muscle biopsies were performed after informed consent was obtained. 2.2. Muscle pathological examination Open muscle biopsies were carried out in all patients. Muscle specimens were frozen in isopentane that was precooled in liquid nitrogen and stored at − 80 °C. For histological examination, serial frozen sections (8 μm) were stained by routine histochemical methods including hematoxylin–eosin and modified Gomori trichrome. For electron microscopic examination, the muscle samples were fixed in 2.5% glutaraldehyde, then in 1% buffered osmium tetroxide, dehydrated in an ascending ethanol series, and embedded in Epon. Semithin sections of muscle were cut and stained with toluidine blue to select muscle fibers for thin sectioning. Thin sections were double-stained with uranyl acetate and lead citrate and then examined under an electron microscope. 2.3. Genetic analysis GNE mutation analysis was performed on all probands, as well as some of their available family members. NM_005476 was used as a reference sequence in this study since the GNE gene was recently reported to have several splice isoforms [1]. Genomic DNA was extracted from peripheral blood or frozen muscle using standard protocols. All exons and their flanking sequences of the GNE gene were amplified by PCR using primers designed by the authors (available on request). After purification, PCR products were directly sequenced using BigDye terminators (Applied Biosystems, Foster City, CA) on an automated sequencer (ABI 3730; Applied Biosystems). Sequencing in the opposite direction was performed whenever an abnormal sequence was found. For those patients with only one heterozygous mutation, we further sequenced the coding exon1 of hGNE2 isoform (NM-001128227) [1]. Novel mutations detected in this study were screened in 100 normal Chinese individuals by direct sequencing of PCR products. Functional prediction was also performed to further confirm the genetic effect of these novel variants. To ascertain the allele frequency of the common GNE mutation p.D176V in the Chinese population, we screened for it in 520 control individuals by direct sequencing of PCR products. Haplotype analysis was performed in 11 patients carrying p.D176V mutation to determine whether this mutation represents a single founder mutation. Single nucleotide polymorphisms (SNPs) at 10 intragenic or flanking loci spanning 40 kb were identified using HapMap (CHB

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database, Hapmap release 28, August 2010). Allele frequencies for these SNPs were between 55:45 and 72:28. Each SNP was amplified by PCR using primer pairs designed by the authors using Primer 3 software (primer sequences are available upon request). Direct DNA sequencing was performed using BigDye terminators (Applied Biosystems) on an automated sequencer (ABI 3730; Applied Biosystems). 3. Results 3.1. Clinical data Clinical data from the 35 patients with GNE myopathy are summarized in Table 1. There were 16 males and 19 females. Seven had an autosomal recessive family history of distal myopathy. The mean age of onset was 30.6 ± 7.3 years, ranging from 20 to 43 years. The interval from disease onset to diagnosis was 1 to 30 years. According to their initial symptoms and distribution of muscle weakness, patients could be divided into two groups: typical distal phenotype and noncanonical clinical phenotype. The 29 patients in the typical group presented with a classic phenotype, that is, weakness of the distal muscles in the anterior compartment of the lower extremities as their initial symptom. The quadriceps was usually spared even years after disease onset. With progression of the disease, both the proximal and distal muscles of the upper extremities, neck flexors, and pelvic muscles developed weakness and muscle atrophy. Physical examination showed weakness of the TA muscles in all cases (MRC grade 0–3), while the strength of their quadriceps femoris was MRC grade 4–5. The remaining six patients belonged to atypical group. Two of them (Patients 17 and 22) initially complained of difficulty climbing stairs, without obvious ankle weakness. They consistently showed more prominent muscle weakness in their quadriceps (MRC grade 3 and 4−, respectively) than in their TA (MRC grade 4 +), and both were classified as having a limb-girdle phenotype. The initial symptoms in Patient 6 were difficulty in lifting her right arm, and weakness of the TA developed one year later. Patient 25 presented with concurrent distal muscle weakness in his upper and lower extremities. While most patients showed symmetrical distal weakness, two cases (Patient 8 and 27) showed notably asymmetrical muscle weakness. Laboratory examination revealed that serum CK level was mildly to moderately elevated in most patients (less than 10 folds of normal limits), except that in one patient it was greater than 10-fold. Electromyography in 32 cases showed myogenic changes in 25, neurogenic changes in 3, and mixed changes in 4. There were 11 typical patients and 2 atypical patients who underwent muscle MRI examination. In the typical group, muscle MRI of legs showed predominant fatty changes in the anterior and superficial posterior compartments of the lower legs, and the posterior and medial compartments of the thigh muscles. The muscles in the deep posterior compartment of lower legs and anterior compartment of thighs were least affected (Fig. 1). Notably, the vastus intermedius, vastus medialis, and rectus femoris could be seriously compromised, but the vastus lateralis could be completely spared or only mildly infiltrated with fatty tissue even 30 years after disease onset. In the atypical group, the pattern of thigh muscle involvement in the patient with limb-girdle phenotype (Patient 22) was roughly similar to that seen in the typical group, except that both vastus lateralis and rectus femoris were relatively spared, and the muscles in the superficial and deep posterior compartments were equally seriously involved while the muscles in the anterior compartment including tibialis anterior and peroneus muscle were mildly affected (Fig. 1). In patient 8, who showed asymmetrical distal weakness clinically, MRI also revealed asymmetrical fatty infiltration in his tibialis posterior, popliteus, flexor digitorum and soleus. Analysis of muscle fatty infiltration of each muscle in all of these 13 patients by using the modified Mercuri scale revealed that those most


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frequently affected by fatty replacement were the adductor longus and semitendinosus, followed by the soleus, semimembranosus, long head of the biceps femoris, anterior tibialis, adductor magnus, and gastrocnemius. The least affected muscles were the vastus lateralis and gluteus maximus (Supplement Figs. 1 and 2). It is noteworthy that although anterior tibialis was often involved, it was not the most seriously involved in both typical and atypical patients. 3.2. Muscle pathology Muscle biopsies were performed in all patients. Rimmed vacuoles were observed in all cases (Fig. 2). Moderate-to-marked variation in fiber size with occasional fiber necrosis or regeneration was observed in some cases. Electron microscopy revealed autophagic vacuoles in all cases. 3.3. Results of genetic analysis Mutation analysis identified that all patients carried GNE mutations (Table 1 and Fig. 3). Five patients were homozygous, with p.D176V in two patients, and c.1525C N T (p.H509Y), c.38G N C (p.C13S), and c.1760T N C (p.I587T) in one each. Twenty-eight patients were compound heterozygous, with 27 different mutations. Two patients had detected only one heterozygous c.1571C N T (p.A524V) mutation site. In total, we detected 29 different GNE mutations in this study. Of these, 16 were novel mutations: c.140G N A (p.G47D), c.197T N A (p.F66Y), c.518A N C (p.E173A), c.556T N C (p.Y186H), c.737G N T (p.R246L), c.787C N T (p.R263*), c.916C N T (p.R306*), c.1097C N A (p.A366D), c.1534G N A (p.V512M), c.1559G N A (p.C520Y), c.1633G N C (p.G545R), c.1642G N A (p.G548S), c.1865T N G (p.V622G), c.1912G N C (p.A638P), IVS2 + 1G N A and c.2112delC (p.A705fs). All of the novel mutations were absent in 200 control chromosomes from healthy Chinese individuals. Functional prediction using SIFT and Polyphen2 software also indicated that all these novel mutations were probably or possibly damaging except p.R246L (Supplement Table 1). As shown in Fig. 3, mutations were located throughout the entire GNE (NM_005476), including both epimerase and kinase domains. Based on the mutation location, 33 patients with homozygous or compound heterozygous mutations could be divided into three groups: EE group (13 patients, homozygous or compound heterozygous mutations located in the epimerase domain), EK group (17 patients, compound

heterozygous mutations located in the epimerase and kinase domains) and KK group (3 patients, homozygous or compound heterozygous mutations located in the epimerase domain). In the EE group, 11 showed typical and 2 showed atypical phenotype. In the EK group, 14 were typical and 3 were atypical. All the KK group patients showed typical phenotype. The age of onset of EE, EK, and KK group was 31.1 ± 7.5, 30.8 ± 7.3 and 27.3 ± 3.8 years, respectively. Among these mutations, p.D176V was the most frequent, which was detected in 65.7% (23/35) of this patient group, giving an allele frequency of 35.7% (25/70). p.A524V was the second most common, with an allele frequency of 8.6% (6/70). We sequenced the region containing the p.D176V mutation in 520 normal controls. A heterozygous c.527A N T mutation was found in only one individual, giving an estimated allele frequency of 0.096% (1/ 1040); in other words, it is rare among Chinese people. After sequencing 10 SNPs surrounding the c.527A N T (D176V) mutation in the 11 patients harboring the p.D176V mutation, we could not construct a common haplotype for them (Supplement Table 2). This indicated that there may be no founder effect for this mutation among mainland Chinese patients with GNE myopathy. 4. Discussion To gain insight into the genetic basis of GNE myopathy among the mainland Chinese, we screened for GNE mutations in a cohort of 35 unrelated patients. All of the patients in this study presented with progressive distal and proximal muscle wasting and weakness beginning in adulthood, with autosomal recessive inheritance or no family history (sporadic disease). Rimmed vacuoles in muscle biopsies, exclusion of other rimmed vacuolar myopathies, and identified GNE mutations, supported the diagnosis of GNE myopathy. In our series of 35 patients, 33 had either homozygous or compound heterozygous GNE mutations and two had a single heterozygous GNE mutation who may have another heterozygous mutation in intronic regions [1]. Among the 29 mutations identified, 16 were novel [12], which has broadened the mutational spectrum of the GNE gene. Notably, the genotype distribution in our patient group was quite different from the study by Li et al. [14]. Since Li's study only included a sample size of six patients, which was much smaller than our 35-patient cohort that came from 15 different provinces of mainland China, the difference in genotype distribution could be attributed to the bigger sample size and geographical patient diversity in our study. The p.L508S mutation,

Fig. 1. Axial T1-weighted MR images in patients with different phenotype. A,B: Patient 26 with typical phenotype, revealed serious fatty replacement in the medial and posterior compartment of thigh muscles and almost all lower leg muscles, with bilateral vastus lateralis relatively spared; C,D: Patient 8 with asymmetrical weakness, revealed asymmetrical involvement of adductor magnus and lower leg muscles; E,F: Patient 22 with limb-girdle weakness, revealed fatty replacement and atrophy of medial and posterior compartment of thigh muscles while bilateral vastus rectus, vastus lateralis, anterior tibialis and extensor digitorum were relatively spared. Asterisks: muscles with serious fatty replacement.



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Fig. 2. Myopathological changes in Patient 17 (A and B) and Patient 22 (C and D). H&E (A and C) and mGT (B and D) staining showed rimmed vacuoles and fiber size variation in both cases. Arrows: rimmed vacuoles.

the most common mutation in Li's GNE myopathy patients, was not present in any of our patients. In contrast, the p.D176V mutation was the most frequently observed in our patients. At least one mutant allele was present in 23 of our 35 patients, giving an estimated allele frequency of 35.7%; this is consistent with the findings by Lu et al. [15]. p.D176V is well known as the second most common mutation in Japanese patients with GNE myopathy [4,16]. The second most common mutation in our patient series was p.A524V, which has also been reported in patients from Thailand, Mexico, South Africa, and France [3,17]. However, the p.V572L mutation, another common mutation in both Japanese and Korean populations [4,8], was detected in only one of our patients, with an allele frequency as low as 1.43% (1/70). All of these data suggest that, while the present group of GNE myopathy patients shares one common mutation with those in other Asian countries, the genotype patterns are not completely consistent. Because p.D176V mutation accounts for one third of the abnormal alleles in our series—indicating a high frequency of carriers in China—we screened for it in 520 normal controls. It was present in only one of these individuals. Thus, the estimated allele frequency of this mutation in the population is 1:1040. According to the Hardy– Weinberg equation, a carrier frequency of 1:520(0.19%) implies that 1:1,081,600 Han Chinese are expected to suffer from GNE myopathy. Celeste et al. estimated the worldwide prevalence of GNE myopathy to be 4–21/1,000,000 based on allele frequencies [12]. Until now, however, there have been no more than 100 Chinese GNE myopathy patients

reported. Considering the vast population of China, this suggests that many patients are undiagnosed. To test whether the high frequency of the p.D176V mutation is because of a founder effect, we performed haplotype analysis in patients with this mutation. Our results showed no founder effect, suggesting that these patients did not share a common ancestor. Similarly, Nishino et al. reported that the haplotypes of two pedigrees with p.D176V mutation were completely distinct from each other [16]. However, considering that p.D176V is a common mutation both in China and Japan, which are geographically close, the possibility of a founder effect cannot be formally excluded. Further studies are needed. Among these 35 unrelated index patients, most showed a clinical picture typical of GNE myopathy, which was similar to that reported in other regions, including young adult onset, preferential involvement of the TA muscle as the initial sign, and mild-to-moderate elevation of serum CK. However, some patients in our series presented with atypical clinical manifestations. First, two presented with proximal lower limb weakness; their muscle weakness was more prominent in the quadriceps than in the TA even 2 to 9 years after disease onset. Second, weakness of the arms was occasionally the initial presentation. Third, a few patients showed asymmetric weakness of the legs. Atypical clinical features associated with GNE myopathy have also been reported by others. Motozaki et al. reported GNE mutations associated with proximal leg weakness and necrotizing myopathy [18]. Recently, Park et al. reported that about half of 11 GNE myopathy patients had initial limb-

Fig. 3. GNE mutation spectrums in 35 Chinese GNE myopathy patients. Sixteen novel mutations are shown in bold.


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girdle weakness that remained throughout the disease course [10]. GNE myopathy can even occasionally present as asymmetric hand weakness [11]. This indicates that the clinical spectrum in GNE myopathy shows great heterogeneity, making the initial diagnosis difficult in atypical patients [19]. Until now the genotype–phenotype correlation in GNE myopathy hasn't been definitely established yet. In our patient cohort, we found that atypical patients had at least one allele mutation in the epimerase domain. Previous studies have also reported that those with typical features of GNE myopathy are associated with homozygous mutations in the kinase domain, while the presence of a mutation in the epimerase domain may develop unusual clinical features [19–21]. The age of onset was a bit younger in the KK group than that of EE and EK groups in the present study, suggesting that there might be correlations between disease severity and mutation location. One recent study in 212 Japanese GNE myopathy patients also showed homozygous p.V572L mutation resulting more severe phenotypes, while p.D176V mutation associated with milder phenotype [4]. Since there were only 5 homozygous patients in our patient cohort, more Chinese patients with GNE myopathy are needed to get convincing data on the genotype–phenotype correlation. In recent years, muscle MRI has proven useful to help diagnose muscle disease. Different diseases show different patterns of muscle involvement. Similar to the findings by Tasca et al. [22], muscle MRI of our patients showed frequent involvement of both the posterior and medial compartments of the thigh muscles, and the anterior and superficial posterior compartments of the lower legs. Among the leg muscles, the soleus and adductor longus were most frequently and seriously involved, followed by the semitendinosus, adductor magnus, semimembranosus, gastrocnemius, and long head of the biceps femoris. The gastrocnemius and soleus could be seriously involved even at an early stage, which may indicate subclinical damage. It is noteworthy that the vastus lateralis (not the entire quadriceps) was the only muscle spared in the advanced stages of GNE myopathy, while the rectus femoris, vastus intermedius, and medialis showed variable degrees of fatty replacement [22]. Even in patients with an atypical phenotype, the vastus lateralis was still relatively spared from fatty changes; this is consistent with a report by Park et al. [10]. The preferential sparing of the vastus lateralis seems to be the unique feature of GNE myopathy and could offer valuable information for differential diagnosis. However, it cannot be concluded that GNE myopathy be diagnosed solely by muscle MRI findings. Other distal or rimmed vacuolar myopathies may demonstrate similar patterns as GNE myopathy at certain stage of disease. For example, desmin myopathy could show more serious involvement of distal muscles than proximal muscles, and peroneous, semitendinosus, sartorius and gracillis are significantly involved with quadriceps muscle relatively spared [23]. The typical muscle imaging pattern of KHLH9 mutated distal myopathy is pronounced involvement of tibialis anterior, gastrocnemius, soleus, semimembrenus, biceps femoris and vastus intermedius with relatively sparing of vastus lateralis and vastus medialis [24]. It's difficult to distinguish GNE myopathy from these myopathies just based on imaging evidence. Besides, at the late stage of most disease, almost all distal and proximal muscles are involved, which creates great difficulty in judging the degree of muscle involvement. Therefore the final diagnosis of GNE myopathy should be based on clinical, muscle imaging findings, myopathological features and GNE mutation analysis. In conclusion, our results showed that p.D176V in the GNE gene was the most common mutation in 35 unrelated GNE myopathy patients from mainland China, followed by p.A524V. The 16 novel mutations we found have broadened the genotypic spectrum of the GNE gene. Most of our patients showed typical features of clinical manifestation and myopathological findings, although some rare symptoms or atypical changes were observed in some patients; muscle MRI findings can provide clues for differential diagnosis of the latter.

Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jns.2015.04.028. Conflict of interest There is no conflict of interest. Acknowledgments This study was supported by the Ministry of Science and Technology of China (No. 2011ZX09307-001-07). References [1] Huizing M, Carrillo-Carrasco N, Malicdan MC, Noguchi S, Gahl WA, MitraniRosenbaum S, et al. GNE myopathy: new name and new mutation nomenclature. Neuromuscul Disord 2014;24:387–9. [2] Eisenberg I, Avidan N, Potikha T, Hochner H, Chen M, Olender T, et al. The UDP-Nacetylglucosamine 2-epimerase/N-acetylmannosamine kinase gene is mutated in recessive hereditary inclusion body myopathy. Nat Genet 2001;29:83–7. [3] Huizing M, Krasnewich DM. Hereditary inclusion body myopathy: a decade of progress. Biochim Biophys Acta 1792;2009:881–7. [4] Cho A, Hayashi YK, Monma K, Oya Y, Noguchi S, Nonaka I, et al. Mutation profile of the GNE gene in Japanese patients with distal myopathy with rimmed vacuoles (GNE myopathy). J Neurol Neurosurg Psychiatry 2014;85:914–7. [5] Sadeh M, Gadoth N, Hadar H, Ben-David E. Vacuolar myopathy sparing the quadriceps. Brain 1993;116:217–32. [6] Broccolini A, Ricci E, Cassandrini D, Gliubizzi C, Bruno C, Tonoli E, et al. Novel GNE mutations in Italian families with autosomal recessive hereditary inclusion-body myopathy. Hum Mutat 2004;23:632. [7] Massa R, Weller B, Karpati G, Shoubridge E, Carpenter S. Familial inclusion body myositis among Kurdish–Iranian Jews. Arch Neurol 1991;48:519–22. [8] Kim BJ, Ki CS, Kim JW, Sung DH, Choi YC, Kim SH, et al. Mutation analysis of the GNE gene in Korean patients with distal myopathy with rimmed vacuoles. J Hum Genet 2006;51:137–40. [9] Nalini A, Gayathri N, Nishino I, Hayashi YK. GNE myopathy in India. Neurol India 2013;61:371–4. [10] Park YE, Kim HS, Choi ES, Shin JH, Kim SY, Son EH, et al. Limb-girdle phenotype is frequent in patients with myopathy associated with GNE mutations. J Neurol Sci 2012;321:77–81. [11] de Dios JK, Shrader JA, Joe GO, McClean JC, Williams K, Evers R, et al. Atypical presentation of GNE myopathy with asymmetric hand weakness. Neuromuscul Disord 2014;24:1063–7. [12] Celeste FV, Vilboux T, Ciccone C, de Dios JK, Malicdan MC, Leoyklang P, et al. Mutation update for GNE gene variants associated with GNE myopathy. Hum Mutat 2014; 35:915–26. [13] Wang ZX, Gao YY, Zhang Y, Bu DF, Yuan Y. Novel mutations in GNE gene in Chinese patients with Nonaka myopathy. J Apoplexy Nerv Dis 2006;23:201–3 [Chinese]. [14] Li H, Chen Q, Liu F, Zhang X, Liu T, Li W, et al. Clinical and molecular genetic analysis in Chinese patients with distal myopathy with rimmed vacuoles. J Hum Genet 2011; 56:335–8. [15] Lu X, Pu C, Huang X, Liu J, Mao Y. Distal myopathy with rimmed vacuoles: clinical and muscle morphological characteristics and spectrum of GNE gene mutations in 53 Chinese patients. Neurol Res 2011;33:1025–31. [16] Nishino I, Noguchi S, Murayama K, Driss A, Sugie K, Oya Y, et al. Distal myopathy with rimmed vacuoles is allelic to hereditary inclusion body myopathy. Neurology 2002;59:1689–93. [17] Liewluck T, Pho-iam T, Limmongse C. Mutation analysis of the GNE gene in distal myopathy with rimmed vacuoles (DMRV) patients in Thailand. Muscle Nerve 2006;34:775–8. [18] Motozaki Y, Komai K, Hirohata M, Asaka T, Ono K, Yamada M. Hereditary inclusion body myopathy with a novel mutation in the GNE gene associated with proximal leg weakness and necrotizing myopathy. Eur J Neurol 2007;14:e14–5. [19] Grandis M, Gulli R, Cassandrini D, Gazzerro E, Benedetti L, Narciso E, et al. The spectrum of GNE mutations: allelic heterogeneity for a common phenotype. Neurol Sci 2010;31:377–80. [20] Chu CC, Kuo HC, Yeh TH, Ro LS, Chen SR, Huang CC. Heterozygous mutations affecting the epimerase domain of the GNE gene causing distal myopathy with rimmed vacuoles in a Taiwanese family. Clin Neurol Neurosurg 2007;109:250–6. [21] Del Bo R, Baron P, Prelle A, Serafini M, Moggio M, Fonzo AD, et al. Novel missense mutation and large deletion of GNE gene in autosomal-recessive inclusion-body myopathy. Muscle Nerve 2003;28:113–7. [22] Tasca G, Ricci E, Monforte M, Laschena F, Ottaviani P, Rodolico C, et al. Muscle imaging findings in GNE myopathy. J Neurol 2012;259:1358–65. [23] Fischer D, Kley RA, Strach K, Meyer C, Sommer T, Eger K, et al. Distinct muscle imaging patterns in myofibrillar myopathies. Neurology 2008;71:758–65. [24] Cirak S, von Deimling F, Sachdev S, Errington WJ, Herrmann R, Bönnemann C, et al. Kelch-like homologue 9 mutation is associated with an early onset autosomal dominant distal myopathy. Brain 2010;133:2123–35.


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