Unusual expression of emerin in a patient with X-linked Emery–Dreifuss muscular dystrophy

Neuromuscular Disorders 10 (2000) 567±571

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Unusual expression of emerin in a patient with X-linked Emery±Dreifuss muscular dystrophy C. Di Blasi a, L. Morandi a, M. Raffaele di Barletta b, S. Bione b, P. Bernasconi a, M. Cerletti a, R. Bono c, F. Blasevich a, D. Toniolo b, M. Mora a,* a

Department of Neuromuscular Diseases, Istituto Nazionale Neurologico `C. Besta', Via Celoria 11, 20133 Milan, Italy b Institute of Genetics, Biochemistry and Evolution, CNR, Pavia, Italy c Department of Pediatric Neurology, Istituto Nazionale Neurologico `C. Besta', Milan, Italy Received 2 July 1999; received in revised form 22 March 2000; accepted 23 March 2000

Abstract We report on a patient with the typical clinical ®ndings of Emery±Dreifuss muscular dystrophy due to a mutation in the emerin gene that should have produced a higher molecular weight protein. Immunohistochemical analysis showed emerin localized only in the cytoplasm of muscle ®bres and lymphoblastoid cells. The emerin molecule contained the nucleoplasmic domain and the transmembrane domain responsible for nuclear membrane targeting, so its incorrect localization and lack of function could be due to abnormal folding resulting in rapid degradation or inability to bind other nuclear proteins. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Emerin; Emery±Dreifuss muscular dystrophy; Nuclear membrane protein; Immunohistochemistry

1. Introduction

2. Materials and methods

Emery±Dreifuss syndrome is a genetically heterogeneous entity characterized by early contractures, slowly progressive muscle wasting and weakness and cardiomyopathy, usually presenting as heart-block with risk of sudden death [1]. Mutations in at least two genes cause Emery± Dreifuss muscular dystrophy (EDMD); the STA gene [2] encoding emerin, is responsible for the X-linked form, and the LMNA gene encoding lamin A/C is responsible for the autosomal dominant form [3]. Emerin, a small protein of 254 amino acids, localizes to the inner nuclear membrane of numerous cell types. Amino acid sequence homology [2], cellular localization [4±6] and biochemical and molecular characteristics [7] suggest that emerin is a member of the nuclear lamin-associated protein family. Several mutations have been identi®ed in the STA gene, most are nonsense and cause complete loss of emerin [8]. A few missense mutations produce a protein of reduced molecular weight and expression [2,6]. We report here on a patient with X-linked EDMD and unusual emerin expression caused by a mutation in the STA gene.

2.1. Case report

* Corresponding author. Tel.: 139-2-2394413; fax: 139-2-70633874.

The patient is a 14-year-old boy who was ®rst seen at our institute at age 6 years for mild weakness, some dif®culty in running and frequent sudden falls while walking. On neurological examination he presented hypotrophic and hypotonic limb muscles, mild weakness of abdominal and distal leg muscles causing dif®culty in walking on heel and rising from the ¯oor. His intelligence quotient (IQ), assessed by the Wechsler preschool scale and the primary scale of intelligence (WPPSI), was moderately reduced (68). CK was about 2.5 normal value, ECG was normal, EMG showed neurogenic potentials suggesting involvement of the second motor neuron. A muscle biopsy showed increased endomysial connective tissue, ®bre size variability, hypertrophic and hypotrophic angulated ®bres, central nuclei and splittings. Numerous large cores were observed on NADH staining; ATPase showed type I grouping; type I ®bres were hypotrophic. Disease progression was very slow. Elbow and Achilles tendon retractions (158), ®rst observed at 9 years, progressively worsened so that the patient underwent operations to lengthen the Achilles tendons at age 11 years and to lengthen elbow ¯exors at 13 years. When the patient was last seen at 14 years neck and trunk ¯exors,

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C. Di Blasi et al. / Neuromuscular Disorders 10 (2000) 567±571

pelvic girdle muscles and distal leg muscles were moderately weak. Cardiac function evaluation by ECG and echocardiogram was still normal. Neurological examination of the patient's mother showed no muscle weakness or joint contractures. 2.2. Methods 2.2.1. DNA analysis Peripheral blood lymphocytes were isolated by centrifugation on Ficoll±Hypaque (Pharmacia, Uppsala, Sweden) from fresh blood; Epstein±Barr virus-transformed lymphoblasts were prepared. Genomic DNA was prepared from transformed cells of the patient and of a normal individual. The DNA was polymerase chain reaction (PCR) ampli®ed, puri®ed and sequenced using primers and conditions previously described [2]. 2.2.2. Immunocytochemistry Muscle samples were frozen in isopentane cooled with liquid nitrogen and stored in liquid nitrogen until use. Emerin was localized immunocytochemically after methanol ®xation on 6 mm thick cryosections or on lymphoblastoid cell smears, using a previously characterized polyclonal antibody [6] or a commercial monoclonal antibody (Novocastra, Newcastle-upon-Tyne, UK) diluted respectively 1:1000 and 1:50 in phosphate-buffered saline (PBS) plus 10% appropriate species serum. Detection of the primary antibody was with a biotin-avidin system (Jackson ImmunoResearch, West Grove, PA). On some sections emerin was simultaneously localized with lamin B2 which was used as a marker of the nuclear envelope. In this case a mixture of anti-emerin polyclonal antibody and anti-lamin monoclonal antibody (Ylem Srl, Avezzano, Italy) (diluted respectively, 1:1000 and 1:500) was applied, followed by biotinylated anti-rabbit IgG (Jack-

son ImmunoResearch), by rhodamine-streptavidin (Jackson ImmunoResearch), and ®nally by ¯uorescein isothiocyanate-labeled goat anti-mouse IgG (Jackson ImmunoResearch). On adjacent sections, as control, the primary antiemerin polyclonal antibody was either omitted or substituted with preimmune serum, or preadsorbed with puri®ed emerin. On some sections lamin A was also localized using a commercial monoclonal (Chemicon International, Inc., Temecula, CA) diluted 1:50. 2.2.3. Electron microscopy Small muscle fragments and lymphoblastoid cell pellets were ®xed with 4% or 1% glutaraldehyde in 0.12 M phosphate buffer, respectively, post-®xed in 2% osmium tetroxide, dehydrated and embedded in Spurr resin. Ultrathin sections were stained with uranyl acetate and lead citrate and examined in a Philips 410 electron microscope. 2.2.4. Immunoblot Emerin was detected on lymphocyte or lymphoblastoid cell lysate by immunoblot as described [6]. Brie¯y, approximately 2 £ 10 6 cells prepared as described above, were resuspended in Laemmli buffer, boiled for 5 min, resolved on 12.5% sodium dodecylsulfate-polyacrylamide gels and electrotransferred to nitrocellulose membranes. The nitrocellulose ®lters were then incubated with the polyclonal or monoclonal anti-emerin antibody followed by alkaline phosphatase-conjugated secondary antibody (Promega, Madison, WI). 3. Results Sequence analysis of the patient's DNA revealed a deletion of a C at position 1817 (C1817del), causing a frameshift

Fig. 1. Immunoblot of emerin in lymphocytes from the patient (lane 2), the patient's mother (lane 1) and a normal control (lane 3). The molecular weight indicators on the right (kDa) were obtained from molecular markers.

C. Di Blasi et al. / Neuromuscular Disorders 10 (2000) 567±571

4 bp before the normal stop codon, which was eliminated. A new stop codon was found 312 bp downstream. The mutation could produce an abnormal protein with a C terminal portion of 102 extra amino acids. DNA from the patient's mother revealed the same mutation. Immunoblots of total proteins from lymphocytes and lymphoblastoid cells of the patient showed a very faint band, positive to emerin polyclonal antibody, of slightly lower than normal molecular weight (Fig. 1), suggesting that the abnormal protein is degraded. No band was detected

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with the monoclonal antibody. Immunoblot of lymphocytes from the mother showed a normal molecular weight band of reduced intensity but not the band of lower molecular weight seen in the patient. Immunohistochemical analysis of emerin in the patient's skeletal muscle with the polyclonal antibody showed absence of the protein in all nuclei; however bright emerin positivity was seen in circumscribed areas of the cytoplasm in many ®bres (Fig. 2b,c). When observed in co-localized emerin-lamin B sections, these positive areas were only

Fig. 2. Immunolocalization of lamin B and emerin in patient's muscle. Co-localization of lamin B (a) and emerin (polyclonal antibody) (c) showing differing distribution of the two proteins. Localization of emerin (polyclonal antibody) on consecutive sections (b,d,f) showed bright positivity in areas of the cytoplasm and patchy positivity on ®bre surfaces (b). There was no signal when antibody was preadsorbed with puri®ed emerin (d) or preimmune serum was used (f). When monoclonal antibody was used (e) emerin was weakly positive in the cytoplasm (£400).

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seldom in correspondence with nuclei (Fig. 2a,c). Patchy immunoreactivity to emerin was also detected on the surface of several ®bres (Fig. 2b). No positivity was found when the anti-emerin polyclonal antibody was preadsorbed with the protein (Fig. 2d), when preimmune serum was used instead of the antibody (Fig. 2f) or when the primary antibody was omitted (not shown). Using the monoclonal antibody, emerin was completely absent from nuclei and stained weakly in the cytoplasm (Fig. 2e). Immunohistochemical detection with the polyclonal anti-

body in the patient's lymphoblastoid cells showed emerin positivity in the cytoplasm tending to aggregate in granules (Fig. 3c), while in controls emerin was mainly on the nuclear surface, although some positivity was also seen in the cytoplasm (Fig. 3d); this was particularly evident when emerin and lamin B were co-localized (Fig. 3a±c (patient) and Fig. 3b±d (normal control)). With the monoclonal antibody the emerin signal in the patient's lymphoblastoid cells had a distribution similar to that observed with the polyclonal, but was weaker and varied from cell to cell (Fig. 3e). Immunohistochemistry of lymphocytes from the patient's

Fig. 3. Co-localization of lamin B (a,b,f) and emerin (polyclonal antibody) (c,d,g) in lymphoblastoid cells from patient (a,c) and control (b,d), and lymphocytes from mother (f,g). Localization of emerin with the monoclonal antibody in lymphoblastoid cells from the patient (e) (£1000).

C. Di Blasi et al. / Neuromuscular Disorders 10 (2000) 567±571

mother showed normal expression of emerin on the nuclear rim of some cells (as expected in a carrier of an X-linked disease); in other cells positivity was either absent or faint (Fig. 3f,g). Since the lymphocytes have a very narrow cytoplasmic rim it was not possible to determine whether positivity was cytoplasmic or nuclear. We were unable to produce lymphoblastoid cells from the mother. Lamin A expression in the nuclei of the patient's muscle ®bres was similar to that of control muscle (not shown). Electron microscopy of the patient's muscle (not shown) revealed a few apoptotic subsarcolemmal nuclei showing chromatin condensation and nuclear membrane disintegration; there was also some streaming of the Z band. The patient's lymphoblastoid cells did not differ ultrastructurally from those of controls (not shown).

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function underline the importance of the C terminal domain and may be due to abnormal protein folding resulting in degradation or inability to bind interacting proteins. The absence of emerin signal at the expected molecular weight on immunoblot may be because the mutated emerin folds abnormally in SDS and is not recognized by the antibody. The extremely faint reduced molecular weight band (with the polyclonal antibody only) may be a breakdown product of the abnormal protein and suggests rapid degradation. Lack of degradation product in the mother suggests possible upregulation of emerin synthesis in the patient leading to detectable protein; this would not occur in the mother. The proposed abnormal folding can also explain the weak immunolocalization with the monoclonal antibody, which is against a short peptide [11] while the protein is still recognized by the polyclonal antibody.

4. Discussion Little is known of emerin function and how its loss selectively affects heart and skeletal muscle. Emerin is a protein of the nuclear envelope, but has recently been reported localized in the intercalated discs of heart. This additional localization suggested that emerin may interact with various proteins and that interactions at the cardiac intercalated discs play a role in cardiac conduction abnormalities in Xlinked EDMD [7]. This localization detected with polyclonal and monoclonal anti emerin antibodies [7,9] was questioned by Manilal et al. [9] who attributed the speci®city of heart and skeletal muscle involvement in EDMD to tissuespeci®c interactions of emerin and lamins at the nuclear envelope. Although emerin is mainly localized at the nuclear rim, subcellular fractionation studies have also shown it to be present in a cytoplasmic membrane-associated fraction [7]. More recent studies [7,10,11] have shown that the main determinant of emerin localization to the nuclear envelope is the C terminal hydrophobic domain. The N terminal nucleoplasmic domain is also involved in nuclear localization, perhaps via binding to nuclear proteins, and precise nuclear membrane targeting requires the presence of the latter half of the nucleoplasmic domain [11]. In our patient, the emerin molecule contains both the nucleoplasmic domain and the transmembrane domain but the protein is incorrectly localized in skeletal muscle and lymphoblastoid cells. Furthermore the patient has the typical clinical ®ndings of EDMD, the abnormal emerin molecule is therefore non functional. The mutation affects the end of the molecule adding 102 new amino acids terminated by a new stop codon. Its incorrect localization and lack of

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