MOLECULAR MEDICINE REPORTS 13: , Received February 21, 2015; Accepted December 18, DOI: /mmr

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1 MOLECULAR MEDICINE REPORTS 13: , 2016 Clinical, pathological and genetic characteristics of autosomal dominant inherited dynamin 2 centronuclear myopathy Abstract. The aim of the present study was to report on a family with pathologically and genetically diagnosed autosomal dominant inherited centronuclear myopathy (CNM). In addition, this study aimed to investigate the clinical, pathological and molecular genetic characteristics of the disease. This pedigree was traced back three generations, four patients underwent neurological examination, two patients underwent muscle biopsy, and eight family members were subjected to dynamin 2 (DNM2) gene mutation analysis. DNM2 mutations were detected in seven family members, of which four patients exhibited DNM2 mutation specific clinical and pathological features. Lower extremity weakness was the predominant symptom of these patients, however, proximal and distal lower extremity involvement was inconsistent. All patients exhibited marked systematic muscle atrophy and various degrees of facial muscle involvement. The patients presented the typical pathological changes of CNM, and their muscle tissues were heavily replaced by adipose tissue, with clustered distribution of muscle fibers as another notable feature. DNM2 CNM patients of this pedigree exhibited heterogeneous clinical and pathological features, providing a basis for further molecular genetic analysis. Introduction XINHONG LIU 1,2, HUAMIN WU 3, JIAN GONG 4, TAO WANG 2 and CHUANZHU YAN 1 1 Department of Neurology, Qilu Hospital of Shandong University, Jinan, Shandong ; Departments of 2 Neurology, 3 Radiology and 4 Digestive System, Tai'an Central Hospital, Tai'an, Shandong , P.R. China Centronuclear myopathies (CNMs) are a group of rare slowly progressive congenital myopathies, which are characterized by centronuclear dysplasia in muscle fibers. Autosomal dominant inherited mutations of the dynamin 2 (DNM2) gene are the cause of 50% of CNM cases (1). Clinical symptoms of DNM2 associated CNM are milder than those of X linked recessive inherited CNM caused by mutations in Received February 21, 2015; Accepted December 18, 2015 DOI: /mmr the myotubularin 1 gene (MTM1) (2). A third gene, associated with autosomal recessive inherited CNM, is bridging integrator 1 (3). Previous studies on DNM2 associated CNM (DNM2 CNM) cases have demonstrated that it can range from severe neonatal onset to mild adult onset, and the severity of the clinical manifestations vary (4 7). The majority of patients exhibit distal or proximal muscle weakness as a first symptom, presenting as general muscle weakness, eyelid drooping, extraocular muscle paralysis, high arch palate, contracture of the Achilles tendon, trismus, and reduction or lack of tendon reflexes, among which the weakness of the distal or lower limbs is most notable (4,8). Disease progression is predominantly slow and few patients develop independent walking difficulties in middle age or restrictive ventilatory disorders (9). Typical pathological features of DNM2 CNM are type I fiber predominance and type I atrophy, increased proportions of radial fibers and central nuclei in the sarcoplasmic reticulum (1). Approximately 100 DNM2 CNM families have exhibited 18 different DNM2 mutations (10). Patients with mutations affecting the intermediate domains of DNM2 usually present with relatively mild clinical features (5,11), while mutations impacting the pleckstrin protein family homology (PH) domain and GED structures result in more serious clinical manifestations (4). To the best of our knowledge, this study is the first to report on a Chinese family with pathologically and genetically confirmed DNM2 associated autosomal dominant inherited CNM. This study aimed to investigate the clinical, pathological and genetic characteristics of this disease, and contribute to further molecular genetic studies of autosomal dominant inherited CNM. Materials and methods Correspondence to: Professor Chuanzhu Yan, Department of Neurology, Qilu Hospital of Shandong University, 107 Culture West Road, Jinan, Shandong , P.R. China E mail: chuanzhuyancn@163.com Key words: autosomal dominant inheritance, dynamin 2, centronuclear myopathy Subjects. Four patients, two males and two females, belonging to a family with three generations of autosomal dominant inherited CNM (pedigree presented in Fig. 1), were investigated. The patients were recruited from the Qilu Hospital of Shandong University (Jinan, China) and presented muscle weakness. All patients underwent physical examination, genetic testing, and two of the patients further provided muscle biopsies. Detailed case history enquiries and clinical examination were conducted by two neurologists. The present study

2 4274 LIU et al: CHARACTERISTICS OF DYNAMIN 2 CENTRONUCLEAR MYOPATHY Table I. Clinical patient data from d=four cases of dynamin 2 centronuclear myopathy. Variable I1 II2 II4 II5 Gender/age Female Male Female Male Age (years) Onset Adolescence Adolescence Adolescence Adolescence Developmental delay Normal Normal Could walk at 2 years old Unknown First symptom Lower extremity Lower extremity Lower extremity Lower extremity weakness weakness weakness weakness Muscle involvement Neck Upper limb Proximal Distal Lower limb Proximal Distal Limb girdle muscular Upper limb Lower limb Facial muscle involvement Eyelid drooping Ophthalmoplegia High vaulted arch Joint contractures Arched feet Curvature of spine Muscle atrophy Tendon reflexes Serum CK 126 Not tested Not tested Not tested EMG Myogenic damage Myogenic damage Not tested Not tested Dysphagia Breathing difficulty CK, creatine kinase; EMG, electromyography., negative; +, positive; +, likely positive. Muscle strength grade: level 0, muscle paralysis, no contraction; level 1, slight contraction of the muscles, but no action; level 2, muscles only function when not subjected to excessive weight; level 3, muscles can carry out actions against gravity but not with added resistance; level 4, able to counteract some resistance against the muscle; level 5, normal muscle strength. was conducted in accordance with the declaration of Helsinki and with approval from the Ethics Committee of Shandong University (Jinan, China). Written informed consent was obtained from all participants. Pathological specimens. Muscle specimens of two patients were obtained from the left biceps by open biopsy. Fresh muscle samples were snap frozen in liquid nitrogen cooled isopentane (Sinopharm Chemical Reagent Co., Ltd., Beijing, China), and 8 µm tissue sections were cut using a cryo ultramicrotome (Leica 1900; Leica Microsystems GmbH, Wetzlar, Germany) at 20 to 25 C. Tissues were then stained with hematoxylin eosin (H&E), modified Gomori's trichrome, NADH tetrazolium reductase, cytochrome c oxidase, succinate dehydrogenase, lactate dehydrogenase, periodic acid Schiff, and oil red O. Dystrophin staining was carried out with the Figure 1. Dynamin 2 associated autosomal dominant central nucleus myopathy family pedigree. Circle, female; square, male; black, affected; white, unaffected. routine use of dynamin N, R, and C monoclonal antibody immunohistochemical staining in order to analyze dynamin protein expression levels in the sarcolemma using an optical

3 MOLECULAR MEDICINE REPORTS 13: , A B C D E F microscope (Olympus BH 2; Olympus Corporation, Tokyo, Japan). Remaining samples were stored at 80 C until further use. All specimens were reviewed and confirmed by two experienced clinical neurologists. Gene sequencing analysis. Based on the DNM2 genome sequence, primers were designed to cover 22 exons, including alternate exons, exon 10b and exon 13b. DNM2 sequences of eight family members were determined and compared with the human gene mutation database ( uk/ac/index.php) by Beijing ACCB Biotech, Ltd. (Beijing, China). Results G H Figure 2. (A and B) I1, H&E staining, visible adipose tissue, muscle fibers distributed in clusters, fiber size is different in muscle tissue, the fibers are homogeneously small, round and polygonal, with no obvious hyperplasia of the endomysium, >90% of the muscle fibers contain a central nucleus. (C) I1, NADH was used to distinguish between two types of fiber. (D I) II2, eldest son of I1 muscle pathology, (D and E) H&E staining, >90% of muscle fibers contain a central nucleus and exhibit visible adipose infiltration between the fibers. (F) NADH staining, typical appearance of fibrin observed. (G) Trophin staining demonstrates the membrane integrity with fat infiltration between the muscle fibers. (H) ATP staining demonstrated type I fiber predominated, however, a number of type II fibers were observed with normal ATP staining. Clinical data. Table I presents the clinical data of four patients. Proband I1 experienced onset of the disease in adolescence, continuing for >30 years. Lower extremity weakness was the first and predominant symptom, which slowly developed and resulted in marked muscle weakness by the age of 30. Systematic muscle atrophy was apparent, rendering the patient wheelchair bound, and was accompanied by double eyelid drooping and facial weakness. Creatine kinase levels were mildly elevated, electromyography (EMG) demonstrated the typical myogenic changes, and a high spontaneous potential was detected (12,13). The children of I1 (II2, II4 and II5) all experienced onset of the disease in adolescence, and also predominantly presented with lower extremity weakness accompanied by light facial muscle involvement and various degrees of systematic muscle atrophy. Symptoms are first identified at ~20 years of age, while they were capable of taking care of themselves and living normal lives. The grandchildren of proband I1, III1 (13 years old), III2 (4 years old), III3 (4 years old) and III4 (5 years old), did not exhibit symptoms of muscle weakness, and their sports activities were the same as healthy children of the same age. However, III3 exhibited general features of congenital muscular disease, including emaciation, narrow face and high vaulted arch. Judging from the combined inheritance mode and clinical symptoms, this family met the criteria of late onset autosomal dominant inheritance. Pathology of muscle biopsies. Following muscle biopsy (Fig. 2), it was demonstrated that muscle tissues of I1 were largely replaced by fat tissues. H&E staining demonstrated that there was a large amount of adipose tissue distributed within the muscle fibers (Fig. 2). The size of the muscle fibers varied. The fibres appeared slightly round or polygonal without notable muscular intimal hyperplasia and with >90% of muscle fibers containing central nuclei (Fig. 2A C). Dystrophin staining indicated intact membrane structures of myofibrils. In addition, the muscles of II2 demonstrated typical CNM pathological changes, including: i) >90% of fibers containing a central nuclei (Fig. 2D); ii) inconsistent size and rounding of muscle fibers; iii) selective type I fiber predominance and type I and atrophy as demonstrated by ATPase staining (Fig. 2H); and iv) radially arranged, wheel like sarcoplasm with central nuclei (Fig. 2F).

4 4276 LIU et al: CHARACTERISTICS OF DYNAMIN 2 CENTRONUCLEAR MYOPATHY Figure 3. Analysis of G A displacement mutation in exon 8 of the dyanamin 2 gene at position 1,106 in patients, I1, II2, II4, II5, III2, III3, III4. Figure 4. Dynamin 2 gene structure. PH, pleckstrin protein family homology domain; GED, GTP enzyme effector domain; PRD, proline rich domain. While there was marked adipose tissue infiltration among the myofibrils (Fig. 2E), dystrophin staining demonstrated intact myofibril membrane structures (Fig. 2G). Mutation screening. Gene sequencing analysis demonstrated that family members Il, II2, II4, II5, III2, III3 and III4 possess a G A substitution mutation at position 1,106 (1,106G>A) of DNM2 exon 8 (Fig. 3), resulting in an amino acid change from arginine to glutamine at position 369 (R369Q) of the DNM2 protein. As this mutation was not detected in normal individuals (10), it is assumed to be causatively associated with the disease. Discussion CNM was first reported by Spiro et al (14) in 1966 and, as the pathological characteristics were similar to those of myotubes in the fetal period, it was designated myotubular myopathy. However, as no arrest of muscle development at the stages of myotube formation was demonstrated as the cause of the disease, the name centronuclear myopathy was regarded to be more appropriate (15). It was later observed that 50% of CNM cases were the result of autosomal dominant inherited DNM2 mutations (1). To the best of our knowledge, this is the first study of a Chinese family with pathologically and genetically confirmed DNM2 associated autosomal dominant inherited CNM. Among the three generations of this family, there were four patients (Il, II2, II4, II5) of which the oldest son (II2) of the proband (I1) and the disease free grandson (III3) demonstrated general characteristics of congenital muscle disease, such as emaciation, narrow face and high vaulted arch. All four patients experienced onset of disease in adolescence or youth, with lower extremity weakness as the first and predominant symptom, gradually involving the upper limbs. As the disease slowly progressed, muscles exhibited various degrees of atrophy, involving upper and lower extremities, while in proximal and distal muscles, the effects were inconsistent, in contrast to an earlier study (16). In other countries, DNM2 CNM patients have a high incidence of eyelid drooping (9/10, 7/11) and ophthalmoplegia (2/10, 5/11) (16). Proband I1 exhibited characteristic eyelid drooping, which was not observed in the other family members, whereas 18 non DNM2 CNM patients presented with eyelid drooping and ophthalmoplegia, indicating that eyelid drooping is not a unique characteristic of DNM2 CNM patients (17). The four patients of this family exhibited facial muscle involvement, marked in proband I1 and milder in her children, indicating that the incidence of facial muscle involvement may be higher in China and possibly a unique feature of Chinese CNM, however, more cases would be required to further elucidate this. None of the four patients of this family indicated joint contractures, scoliosis, arched feet, or other features that are regarded as characteristic of CNM (18). Mandibular muscle contracture was reported as an important feature of newborn or childhood DNM2 CNM in an Italian study (19). In this pedigree, no breathing disorders were present, although emphasis has been placed on respiratory distress problems in DNM2 CNM patients. Recently, Italian and German CNM studies demonstrated various degrees of restrictive ventilatory disorders (7 10,20). The four patients in the present study demonstrated mental retardation but no dysphagia or diplopia, no associated muscle pain or sensory disorders, however, all exhibited diminished tendon reflexes and muscle tone. Proband I1 and her eldest son exhibited a normal muscle enzymogram, and their EMG exhibited myogenic changes, consistent with previous studies (17,19,21). As current clinical studies on myopathy are not systematic or common, it was difficult to conduct a comprehensive comparative study on a whole family, requiring in depth investigation and follow up domestically and abroad. The diagnosis of the disease depends on muscle pathology, and typical patient manifestations include wide range nuclear ingressions of a large number of muscle fibrous nuclei, a markedly increased proportion of central nuclei, radial arrangement

5 MOLECULAR MEDICINE REPORTS 13: , of the perinuclear muscle mass, type I fiber predominance and type I atrophy. The muscle tissues of proband I1 and her eldest son, II2 exhibited typical CNM pathological changes. Central nucleus pathological manifestations can be found in numerous diseases, including muscular dystrophy, recycled fiber, denervation, and metabolic and toxic myopathy, thereby not defining a specific disease. Within these myopathies, perinuclear sarcoplasmic radial arrangement and type I fiber predominance and type I atrophy would not appear, and these symptoms are characteristic of the pathology of the muscular biopsies of patients with CNM. Therefore, the reliability of pathological diagnosis towards CNM was higher. However, congenital myotonic dystrophy and X linked genetic myotubular myopathy exhibit similar clinical and pathological characteristics and should therefore be differentially diagnosed by genetic analysis of the children and their mother's EMG. Proband I1 presented at a late stage, with muscle tissues heavily infiltrated by adipose tissues, while the dystrophin staining indicated that this fatty infiltration occurred outside of the muscle fiber membrane. In a previous report on CNM patients with extensive pseudo muscular hypertrophy, computerized tomography scanning, ultrasound and histochemical staining demonstrated that this muscular hypertrophy was due to the marked infiltration of muscular bundles by adipose tissues (21). By contrast, patients in the present study did not exhibit muscular hypertrophy but muscular atrophy, pathologically indicated by markedly reduced numbers of muscle fibers. A previous study observed extensive muscular hypertrophy in two patients from one family and divided CNM into two clinical subtypes accordingly, assuming that the subtype with the extensive muscular hypertrophy would rapidly progress. However, their biopsy specimens, regardless of muscle hypertrophy, exhibited no increase in adipose and connective tissues (22), thereby not providing a pathological basis for the connection to muscle hypertrophy and fat infiltration. To verify whether the muscular pathological features of patients in the present study were the result of disease progression or of other specific changes requires further investigation. The DNM2 gene located at 19p13.2 contains 22 exons and five structural domains (Fig. 4), including a GTP enzyme domain (GTPase), an intermediate domain, a PH domain (substrate of platelet leukocyte C kinase), a GTP enzyme effector structural domain, and a structural proline rich domain. The encoded protein is functionally involved in cellular endocytosis, membrane anchoring, actin assembly and centrosome binding. Furthermore, it was observed that the exon 8, mutations 1,105C>T, 1,102G>A and 1,106G>A, in addition to the exon 11 mutation 1,393C>T, were high frequency mutations of this disease, resulting in amino acid changes R369W, E368K, R369Q and R465W, respectively (10). Böhm et al (10) investigated 100 pedigrees in 2012 and identified 18 different mutations of dynamin 2 (DNM2, 19p13.2) with five domains involved. Sequencing results in the present study demonstrated that seven family members (I1, II2, II4, II5, III2, III3 and III4) exhibited the G A substitution mutation at exon 8 position 1,106 of the DNM2 gene, resulting in an amino acid change from arginine to glutamine at position 369 (R369Q) of the DNM2 protein. Bitoun et al (6) reported the 1,106G>A mutation in a French pedigree, where it was not observed in normal individuals, indicating that it was the disease causing mutation. While III2 (4 years old), III3 (4 years old), and III4 (5 years old) carried this DNM2 gene mutation, due to their young age they did not exhibit notable signs of muscle weakness, consistent with the age of onset of the disease in their family. However, III3 exhibited general characteristics of CMD, including emaciation, narrow face and high vaulted arch. In conclusion, a DNM2 CNM pedigree was confirmed pathologically and genetically, and was identified to be consistent with the clinical and pathological changes in previously reported cases. While four patients had facial muscle involvement, this was not accompanied by eyelid drooping and paralysis of extraocular muscles, which is possibly a unique feature of Chinese CNM patients, but requires further investigation into a large number of Chinese cases. Muscle pathologies predominantly consisted of: Muscle fibers exhibiting central nuclei; perinuclear muscle mass demonstrating radial arrangement; type I fibers, and atrophy. In addition, two patients demonstrated massive adipose tissue infiltration, which may be the result of the development of the disease or other specific changes, this requires verification in a large numbers of cases. The sequencing results of the present study demonstrated that seven members of this pedigree carried the G A substitution mutation in exon 8 position 1,106 of the DNM2 gene, resulting in an amino acid change from arginine to glutamine at position 369 (R369Q) of the DNM2 protein, previously indicated as the disease causing mutation (6). While family members, III2 (4 years old), III3 (4 years old), and III4 (5 years old) also exhibited this DNM2 gene mutation, they were probably too young to exhibit notable signs of muscle weakness, which would be consistent with the age of onset of the disease in their family. The results of the present study suggest that careful examination of clinical and pathological features allow for improved molecular genetics analyses in CNM. 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