Heterozygous mutations affecting the epimerase domain of the GNE gene causing distal myopathy with rimmed vacuoles in a Taiwanese family

Clinical Neurology and Neurosurgery 109 (2007) 250–256

Heterozygous mutations affecting the epimerase domain of the GNE gene causing distal myopathy with rimmed vacuoles in a Taiwanese family Chun-Che Chu, Hung-Chou Kuo, Tu-Hsueh Yeh, Long-Sun Ro, Shyue-Ru Chen, Chin-Chang Huang ∗ Department of Neurology, Chang Gung Memorial Hospital and University, 199 Tung-Hwa North Road, Taipei, Taiwan Received 3 May 2006; received in revised form 25 September 2006; accepted 28 September 2006

Abstract Objectives: Studies of distal myopathy with rimmed vacuoles (DMRV) revealed that most patients had mutations in the UDP-Nacetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) gene. However, the correlation between GNE mutations and clinical features was not fully understood. Purposes: To report the correlation between the clinical features and genetic analysis of DMRV patients. Patients and methods: The clinical presentations, histopathological findings, image studies, and genetic analyses of two patients with DMRV from a Taiwanese family were studied. Results: Two compound heterozygous mutations, Ile 241 Ser and Arg 246 Gln, located in the epimerase domain, were identified in both patients, who were of the same generation. In addition, the elder sister showed a progressive muscular dystrophy course with severe quadriceps and trunk muscle involvement. Conclusion: The compound heterozygous mutations in the epimerase domain of the GNE gene are important in the severe phenotype of DMRV. However, the mechanisms leading to this phenotypic heterogeneity still remain to be elucidated. © 2006 Published by Elsevier B.V. Keywords: Distal myopathy; Rimmed vacuole; DMRV; GNE gene; Heterozygous mutation

1. Introduction Adult onset distal myopathy is a primary muscle disorder characterized clinically by progressive muscle weakness and atrophy, beginning in the hands or feet, and pathologically by myopathic changes in the skeletal muscles [1]. Distal myopathy with rimmed vacuoles (DMRV) is characterized by the distinct clinical features of: (1) an autosomal recessive or sporadic disorder with an early adult onset, (2) an involvement of the tibialis anterior muscle with a sparing of the quadriceps muscles, (3) rimmed vacuoles, particularly abundant in atrophic fibers, and the nucleus occasionally containing tubulofilamentous inclusions in muscle biopsies, and

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0303-8467/$ – see front matter © 2006 Published by Elsevier B.V. doi:10.1016/j.clineuro.2006.09.008

(4) a normal or mild elevation of the serum creatine kinase level [2,3]. Hereditary inclusion body myopathy (HIBM), originally called “a vacuolar myopathy sparing the quadriceps”, shares characteristic clinical and histopathologic features similar to DMRV, but has been reported mainly from Middle Eastern countries [4]. Both diseases were mapped to the same region on chromosome 9 [5]. The UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) gene, a bifunctional enzyme, has been found in both HIBM and DMRV patients [6,7]. Thus, these two disorders are thought to be the same disease entity. In previous studies, homozygous mutations were shown to be common in Japanese patients with V572L mutations and Middle Eastern Jewish patients with the M712T mutation [5,8]. However, compound heterozygous mutations have been found in other ethnic groups [6,9,10]. Interestingly, HIBM patients carrying homozygous M712T mutations

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appeared clinically to have typical HIBM with a sparing of the quadriceps muscles, similar to Japanese DMRV patients with homozygous V572L mutations [5,8]. In addition, a marked weakness of the hands and involvement in the proximal muscles, including the quadriceps muscles in heterozygous mutation were also found in Japanese patients and nonJewish HIBM patients [5,11,12]. Therefore, the pattern of muscle involvement was proposed to be mutation dependent [10]. However, in a recent study, different mutations in GNE resulted in different enzyme activities, but not in different

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disease phenotypes in DMRV [13]. In this study, we report the clinical and genetic analyses of two patients with DMRV from a Taiwanese family with prominent quadriceps involvement and heterozygous mutations in epimerase domain.

2. Patients and methods We examined two patients of the same generation from a Taiwanese family, who were affected with DMRV.

Fig. 1. Muscle T1-weighted magnetic resonance imaging (MRI) of patient II-1 examined at the age of 36, showed (A) prominent atrophy and fatty infiltration in the thigh muscles, including the quadriceps (arrows) at the middle thigh level, (B) severe atrophy and fatty replacement, including the gastrocnemius (black arrows) and tibialis anterior (open arrows) muscles at the distal leg level. Muscle MRI study of patient II-3 examined at the age of 24, showed (C) fatty infiltration in the adductor muscles (asterisks) and anterior tibialis muscles (open arrows) with preservation of the quadriceps muscles (arrows).

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2.1. Patient II-1 A 38-year-old woman noticed a steppage gait at the age of 21 years. She had normal mental and physical development, but family history revealed that her younger brother (patient II-3) had a similar disease. Four years later, difficulty in arising from the sitting position and bilateral hand weakness developed. Neurological examinations showed muscle wasting in all four extremities, especially in the distal limbs, including intrinsic hand muscles 4/5 and foot muscles 3/5, as delineated by the Medical Research Council of Great Britain (MRC). Difficulty walking on the heels and toes was noted. Proximal muscle strength also showed weakness in the quadriceps, hamstring and iliopsoas muscles (3-4/5 MRC). Tendon reflexes were generally hyporeflexia. Biochemical analyses were normal except for creatine kinase (294 U/L, reference: 15–130 U/L). The nerve conduction study was essentially normal. Electromyography disclosed fibrillations and positive sharp waves in the tibialis anterior and gastrocnemius muscles, but motor unit potentials were normal. She had become unable to stand and walk without support at the age of 26, and was confined to a wheelchair at the age of 30. She could no longer turn over on the bed at the age of 32. Recently, muscle wasting in the intrinsic hand muscles was noted. Muscle strength was 0/5 at the lower limb muscles, including the quadriceps, and 3/5 at the upper limb muscles, using the MRC scale. The muscle strength of the trapezius and sternocleidomastoid, neck flexors and trunk muscles was also decreased. Magnetic resonance imaging (MRI) of the skeletal muscles showed generalized muscle atrophy with fatty infiltration in the extremities, and the paraspinal, abdominal, and intercostal muscles. Fig. 1A and B shows prominent atrophy and fatty infiltration in the thigh and distal leg muscles in the T1-weighted muscle MRI. 2.2. Patient II-3 A 28-year-old man, the younger brother of patient II-1, was referred to our hospital because of distal leg weakness and muscle wasting, beginning 1 year prior to his referral. He first noted difficulty running at the age of 18. Clinical examinations showed distal leg weakness and muscle atrophy in the tibialis anterior, and decreased tendon reflexes in ankle jerks. Laboratory tests revealed an elevation of serum creatine kinase, 384 U/L (reference: 15–130 U/L). The electrocardiogram showed an incomplete right bundle branch block. On needle electromyography, fibrillations and positive sharp waves were noted in the tibialis anterior and biceps muscles, and early recruitment in the upper and lower limb muscles. MRI of the skeletal muscles (Fig. 1C) disclosed fatty infiltration in the adductor muscle group and the anterior compartment of legs with the preservation of quadriceps muscles. Table 1 shows the clinical manifestations of the two patients with DMRV.

Table 1 Clinical and laboratory findings of distal myopathy with rimmed vacuoles in two Taiwanese patients Patient II-1

II-3

Sex/age (yr) Age at onset (yr) Inherited pattern Initial presentation

Female/38 21 AR Foot drop

Male/28 18 AR Foot drop

Age of disability (yr) Walks with assistance Confined to wheelchair Difficulty in turning over

26 28 32

28 No No

Muscle strengtha Neck flexor muscles

3

5

Upper limbs Deltoid Biceps Triceps Intrinsic hand muscles

3 3 2 2

4 4 3 4

Lower limbs Quadriceps Hamstrimgs Tibialis anterior Gastrocnemius

1 1 0 0

5 4 1 3

294

384

Diffuse rimmed vacuoles, atrophy with variation of fiber sizes from the gastrocnemius (24)

Normal from the vastus lateralis (24)

34 +++ ++++ ++++ ++++

24 – ++ ++ +

Creatine kinse (reference: 15–130 U/L) Muscle biopsy (age of biopsy)

Muscle MRIb Age of examination (yr) Quadriceps Hamstrings Tibialis anterior Gastrocnemius

AR: autosomal recessive; yr: years; ‘+’ indicates 0–25% of muscle fibers with fatty infiltration; ‘++’ indicates 25–50% of muscle fibers with fatty infiltration; ‘+++’ indicates 50–75% of muscle fibers with fatty infiltration; ‘++++’ indicates 75–100% of muscle fibers with fatty infiltration. a Muscle strength by the Medical Research Council of Great Britain. b Muscle MRI: muscle magnetic resonance imaging.

2.3. Histopathology, electron microscopy and immunohistochemistry Muscle biopsies were obtained from the gastrocnemius of patient II-1 and the vastus lateralis of patient II-3 after informed consent was obtained to understand subclinical involvement of the quadriceps muscles. The muscle specimens were frozen in cooled isopentane, and stored at −70 ◦ C until analysis. Cryostat sections of 7 ␮m thickness were prepared and stained with hematoxylin and eosin (H&E), modified Gomori-trichrome, NADH-TR, succinate dehydrogenase, and serial ATPase at pH 4.3, 4.6, and 9.5. For electron microscopic (EM) examination, muscle specimens were prepared as previously described [14]. Immunohistochemistry studies, including dystrophin (DYS1: rod domain, DYS2:

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C-terminal domain, DYS3: N-terminal domain), ␣-, ␤-, ␥-, and ␦-sarcoglycan, ␤-dystroglycan, laminin ␣2 and dysferlin were prepared for identifying the deficiency of proteins in the sarcolemmal areas. 2.4. DNA analysis DNA was extracted from the peripheral blood leukocytes using a DNA extraction kit (Stratagene). This study was approved by the institutional review board of Chang Gung Memorial Hospital, and informed consent was obtained from each member of the family. Eleven coding exons (exons 2–12) and flanking sequences of the GNE gene were amplified by polymerase chain reaction (PCR), using primers and conditions as previously described [5,8]. The PCR products were subjected to sequencing using an automated DNA sequencer (MegaBACE Analyzer).

3. Results 3.1. Histopathology, electron microscopy and immunohistochemistry The findings of the specimens from patient II-1 included a markedly increased variation of muscle fiber diameter (15–145 ␮m in diameter), proliferation of connective tissues in the endomysium, fatty replacement, fiber splitting and the presence of internal nuclei in the H&E staining. Staining for modified Gomori-trichrome revealed many rimmed vacuoles in some atrophic fibers (Fig. 2A). In patient II-3, there was no evidence of myopathy under light microscopy (Fig. 2B). EM studies from patient II-1 (Fig. 2C) showed some disorganized muscle fibers and a broad spectrum of non-specific alterations. No typical intracytoplasmic microfilaments and dense bodies in rimmed vacuoles were found. The immunoactivities of antibodies against dystrophin, sarcoglycan, laminin ␣2 , ␤-dystroglycan, and dysferlin were normally expressed in the sarcolemma of both patients when compared with reference control with steroid myopathy (Fig. 3).

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3.2. Haplotype analysis The family members were genotyped using polymorphic markers linked to the GNE gene, revealing the chromosome segregation with the disease (Fig. 4). Haplotype analysis showed that the two affected siblings, II-1 and II-3, had inherited the same chromosome from their parents, indicating that the disease phenotype was linked to markers flanking the GNE gene. 3.3. Detection of the GNE gene mutation Using direct nucleotide sequence analysis of the PCR products covering all 11 exons of the GNE gene, we identified 2 mutations in these 2 patients. The mutations included a G to T transition at coding sequence 773 in exon 4, substituting isoleucine for serine at amino acid position 241 (Ile 241 Ser), and an A to G transition at coding sequence 788 in exon 4, converting arginine to glutamine at codon 246 (Arg 246 Gln) (Fig. 5). The missense mutations were heterozygous, and were not observed in 50 healthy Taiwanese subjects. Both patients, II-1 and II-3, inherited the Ile 241 Ser mutation from their mother (I-2), while the Arg 246 Gln mutation was inherited paternally (I-1).

4. Discussion In this study, we report two GNE heterozygous missense mutations, I241S and R246Q, in two Taiwanese patients from the same generation, with the typical clinical and histological features of DMRV. Patient II-1 showed a severe progressive pattern with an involvement of the lower legs, including the quadriceps, hamstring, arm, and trunk muscles, and was confined to a wheelchair within a few years after disease onset. Patient II-3, the younger brother, manifested a nearly normal function in the upper limbs and could still walk with mild assistance. In both patients, the quadriceps muscle was well preserved initially. GNE is a dual-function enzyme catalyzing two initial steps in the biosynthesis of the N-acetylneuraminic acid—a

Fig. 2. (A) Muscle biopsies from the gastrocnemius of patient II-1 at the age of 24 showed numerous rimmed vacuoles and an increased variation of muscle fiber diameter (Gomori-trichrome stain 200×). (B) Muscle biopsies from the vastus lateralis of patient II-3 at the age of 24 showed a normal pattern finding (Gomori-trichrome stain 200×). (C) Electron microscopy from patient II-1 showed disorganized of muscle fibers and some vacuoles with non-specific changes (original magnification 3000×).

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Fig. 3. Immunohistochemial analyses of dystrophin, ␣-, ␤-, ␥-, ␦-sarcoglycan, laminin ␣2 , ␤-dystroglycan and dysferlin in the biopsied vastus lateralis muscles of patient II-3 when compared with a control individual of 35 years of age. Normal immunoreactive patterns were all seen in the sarcolemma of the patient II-3 and a reference control (bar = 50 ␮m).

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Fig. 4. The DNA markers linking to the GNE gene, D9S1788, D9S1804, and D9S1791 (from top to bottom) are shown in each member of a Taiwanese family.

precursor of sialic acid [15]. Sialic acid modifies the glycoprotein and glycolipids expression at the cell membrane and plays a crucial role in cell adhesion and signal transduction. Although GNE is the rate-limiting enzyme in the sialic acid biosynthetic pathway [16,17], the process by which HIBM or DMRV mutations in the enzyme lead to the disease is not fully understood [13]. To date, more than 40 different GNE mutations in DMRV or HIBM have been reported to cause the disease although not all DMRV or HIBM are due to mutations of the GNE gene [18]. Homogyzous M712T is the most common mutation in Middle Eastern Jews [6], and homogygous V572L among Japanese patients [15,16]. In many other ethnic groups, compound heterozygous mutations have been reported [6,9,10,19] however, the phenotypes were not described clearly. The heterozygous mutation, I241S found in our patients has also been reported recently in a Chinese family [20], and the other mutation, R246Q, has been reported in a Bahamian

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family [6]. Both of these compound heterozygous mutations were located in the epimerase domains. With respect to genotype–phenotype correlations, clinical variations were noted when patients had the same mutations. Typical clinical features of quadriceps-sparing myopathy in DMRV and HIBM, were reported in patients with a homozygous mutation in the kinase domain, such as a homozygous V572L change [5–8,18]. In several studies, severe involvement of the hands or quadriceps muscles was noted in patients with heterozygous mutations with at least one mutant allele in the epimerase domain [9–12]. Therefore, the pattern of muscle involvement was reported to be mutation dependent [10]. The marked weakness of the lower limbs and moderate weakness of the upper limbs and trunk muscles noted in our patient II-1 with a compound heterozygous mutation in exon 4 seemed to be consistent with the above suggestions. However, most GNE mutations affecting the epimerase domain are also reported in patients with typical quadriceps-sparing HIBM. Despite the identification of the GNE gene mutations, we still do not fully understand how these mutations contribute to the pathophysiology of DMRV/HIBM. The two domains of the GNE protein have been reported to catalyze the enzymatic reactions separately and independently. Noguchi et al. [21] demonstrated that the levels of sialic acid in muscle and primary culture cells from DMRV patients were reduced to 60–75% of those of the controls. Saito et al. [22] described a decreased lectin reactivity of skeletal muscle glycoproteins in one case of DMRV with compound heterogenous mutations of both alleles in the epimerase domain, and suggested that hyposialylation of glycoproteins may be involved in the pathogenesis of muscle dysfunction of DMRV patients. Recently, Penner et al. [13] reported that mutations in the epimerase domain were mainly located at the protein surface, and mutations in the kinase domain were localized inside the enzyme. In addition, mutations may influence the function of the domain that does not harbor them. The researchers also concluded that HIBM mutations in GNE cause reduced functionality of the enzyme and may result in a reduced level of sialation, and the differences in the age of onset or in the progression of the disease are most likely due to individual variations. Furthermore, Sparks et al. [23] also revealed that mutations in one enzymatic domain affected not only

Fig. 5. Sequence analysis of polymerase chain reaction products amplified from genomic DNA of a patient with distal myopathy and rimmed vacuoles (patient II-1). The T to G transversion at nucleotide 773 in exon 4 results in a conservative amino acid change (Ile 241 Ser), and the G to A transition at nucleotide 788 in exon 4 results in a missense mutation (Arg 246 Gln).

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that enzyme activity, but also the activity of other domains in GNE. Therefore, further investigation into the correlations among genotypes, enzyme activities and phenotypes are warranted.

Acknowledgements The authors would like to thank Ms. Yu-Chen Hsieh for typing the manuscript. This work was supported by grants from the National Science Council of Republic of China (NSC 89-2314-B-182A-217) and the Neuroscience Research Center (CMRPG34008).

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