THE SPECTRUM OF CONGENITAL MYOPATHIES IN ROMANIA – A PATHOLOGICAL RETROSPECTIVE STUDY

Objectives. Congenital myopathies (CM) are a highly heterogeneous group of disorders with genetic cause, characterized by motor defi cit and weakness usually manifesting in the neonatal period, with slowly progressive or non-progressive course and affecting both sexes. MC classifi cation has undergone many changes over time, and in recent years molecular genetic studies have enabled identifi cation of novel genes and mutations, thus increasing the diagnostic complexity. We wanted to study the incidence and morphological features of the CM cases diagnosed by muscle biopsy in the Pathology Department of Colentina University Hospital over a period of 10 years (09.200509.2015). Materials and methods. We retrospectively reviewed all the muscle biopsies diagnosed with different types of CM. Muscle biopsies were performed and specifi cally processed using routine and special stains on cryosections, semithin and ultrathin sections for ultrastructural examination. In all the cases we reassessed the clinical and laboratory data. Results. From a number of 1,530 peripheral nerve and muscle biopsies performed and analyzed in the 10 years period we diagnosed CM in 15 cases, representing 1.03% of the total. Of these, fi ve were “central core myopathies”, fi ve centronuclear/myotubular myopathies, one case of nemaline myopathy, one case of “reducing body myopathy” and three cases with congenital fi ber type disproportion. Reassessment of morphological data in the clinical context allowed us to identify numerous overlaps between subtypes both in the clinical and pathological picture Conclusions. The reduced number of MC identifi ed in our country suggests that these diseases are probably underdiagnosed or diagnosed late, requiring a better understanding of the various clinical and pathological particularities. In the accurate diagnostic algorithm, muscle biopsy remains essential to establish the type of CM and thus to direct genetic tests.


INTRODUCTION
Congenital myopathies (CM) are rare inherited primary disorders of musculature causing generalized weakness distributed more often proximal, presenting often in the neonatal period, hypotonia expressed in the clinical picture of fl oppy child and often delayed motor development in the fi rst years of life, resulting in death due to cardiorespiratory complications. In other cases, the onset of clinical features is late during childhood or even in adulthood, and in such situations the life span may be close to normal. In most of the CM the inheritance is autosomal recessive or autosomal dominant, so both sexes are nearly equally affected. X-linked forms are also encountered and are manifested in affected boys and symptomatic female carriers. De novo mutations are common as well. Most of the forms are clinically stable, while others have a slowly progressive course. The morphological features of CM were described with the advance of histochemical, enzyme histochemical and ultrastructural techniques and further development of molecular genetics identifi ed the etiology of many types of CM (Table 1). Many mutations in different genes encoding for muscular structural proteins were described in the last years and were associated with specifi c patterns of muscular abnormalities, but there are other CM with yet unknown genetic defects. Another important issue is, like in many other genetic muscle diseases, that genotype-phenotype correlations are complex: a specifi c subtype of CM is due to mutations in many genes, while a different muscular pathology may be associated with mutations in the same gene. Furthermore, even the underlying pathophysiological mechanisms are still debated (1)(2)(3).
The International Standard of Care Committee for Congenital Myopathies published in 2014 a consensus statement providing updated guidelines for the physicians involved in diagnosing CM (1). Based on these recommendations, we aimed to assess the relative incidence and spectrum of CM subtypes in the Romanian pediatric and adult population, based on the pathological aspects identifi ed on the muscle biopsies of these patients. Even now, with increased application of next generation sequencing techniques, the subtypes of CM are still defi ned on the basis of characteristic histopathological features.

MATERIALS AND METHODS
We retrospectively analysed the cases morphologically assessed over a ten years period at the Pathology Department of Colentina University Hospital in the laboratory for neuromuscular pathology. We selected and analyzed the muscle biopsies of all cases previously diagnosed as CM. The skeletal muscle biopsies were performed in our Department of Surgery under local anaesthesia, by a trained surgeon and the myopathologist, after informed consent was obtained from all patients or, in children, from their parents. The selection of the muscle to be biopsied was made by the neurologist/ pediatric neurologist after a detailed clinical examination, sometimes followed by imaging studies, in order to choose a moderately affected muscle, thus avoiding to take the biopsy from a severely involved muscle with end-stage pathology. The samples were oriented immediately after removal and fresh frozen in isopentane precooled in liquid nitrogen at -160 0 C and stored at -80 0 C. 7 μm thick serial muscle cryosections were subjected to routine and special stainings: haematoxylin-eosin (HE), modifi ed Gomori trichrome, van Gieson (GT), Sudan Black B, Periodic acid Schiff (PAS), succinic dehydrogenase (SDH), lactic dehydrogenase (LDH), nicotinamide adenine dinucleotide tetrazolium reductase (NADH), cytochrome c oxidase (COX), adenosine triphosphatase -ATPase pre-incubated at pH 9.4, 4.63 and 4.35. Immunohistochemical stains for some muscle proteins had been performed also on cryosections in certain cases or for differential diagnosis. Another piece of muscle tissue had been preserved in glutaraldehyde and embedded in resine for further ultrastructural analysis, essential in CM cases. A part of the sample had been formalin fi xed and embedded in paraffi n for conventional histopathological processing and routine stainings. We retrieved all the clinical data, including family history, age at presentation, pattern of muscle weakness and disease progression, extramuscular signs, level of creatine phosphokinase (CK) and electromyogram fi ndings.

RESULTS
In a period of ten years (September 2005 -September 2015), out of 1,530 patients reffered to us for muscle/muscle and sural nerve biopsy we diagnosed a total of fi fteen cases of CM of different subtypes: central core disease, nemaline myopathy, centronuclear/myotubular myopathies, congenital fi ber type disproportion and reducing body myopathy.

Central core disease
Among the analyzed cases we identifi ed fi ve cases of central core disease: one child and four adults. The morphological diagnosis was based on the presence of cores, defi ned as single or multiple extensive areas in most of the muscle fi bres that are devoid of oxidative enzyme activity, centrally or peripherally located, caused by absence of mitochondria and sarcoplasmic reticulum in the core region ( Fig. 1) (4). Cores were best highlighted on SDH, LDH, NADH and COX stains.
Another important aspect was type I fi bre uniformity, which may occur even in the absence of cores. Fibrosis and adipose metaplasia were prominent features in cases with long history of symptoms, thus resembling a congenital muscular dystrophy, a diagnosis that had to be ruled out. One of the patients was a very hypotonic child aged 1 year and 4 monts, with the appearance of fl oppy child and life-threatening breathing problems from birth with respiratory failure. His left quadriceps muscle biopsy revealed the presence of diagnostic cores in more than 80% of the muscle fi bers. Another case was a 25 years old man with fatigability noted fi rst at the age of 10 years, atrophy of the distal musculature in the lower limbs, and diffi culties in climbing stairs. The clinical suspicion was a form of limb-girdle muscular dystrophy. CK level was always in the normal limits and EMG was mildly myopathic. On the left deltoid muscle biopsy ( Fig.  1) we identifi ed a variation in the fi ber size, almost only type I fi bers on ATP-ase stains and numerous excentric cores and therefore diagnosed him with central core disease.
The genetic test subsequently performed in France confi rmed our diagnosis and proved a de novo p.Arg4861His (c.14582G>A) mutation on RYR1 gene, which was not detected in his parents. The mutation was located in the transmembrane domain of the calcium channel.

Nemaline myopathy
Nemaline myopathy is characterised morphologically by the presence of nemaline bodies consisting of proteins derived from the Z disc and thin fi lament (5,6). In the last ten years we only diagnosed one case of nemaline myopathy in a boy of 15 years with no family history of muscle diseases. He had muscle weakness and hypotonia since early childhood predominantly in facial muscles, facial dysmorphisms with long face, a tent shaped mouth, high arched palate, also weakness of cervical and trunk fl exor, foot dorsifl exor and fi nger extensor muscles. Furthermore, the patient had respiratory insuffi ciency, dysphagia, foot deformities, scoliosis, chest deformities, superior and inferior limbs contracture and had a waddling gait. CK level was slightly elevated and EMG was myopathic. The pediatric neurologist made the clinical diagnosis of probable congenital myopathy, suspecting a nemaline myopathy on clinical signs and ordered a muscle biopsy from the right gastrocnemius, showing numerous subsarcolemmal and intermyofi brilar nemalinic rods, looking like dark red-blue structures identifi ed on modifi ed Gomori trichrome stain and uniformity of type I fi bers. The rods could be identifi ed in the cytoplasm but not also in the nuclei. The mean diameter of the muscle fi ber was under the normal limits for the age and there was a severe increase in the interfascicular collagen. The presence of rods in over 50% of the muscle fi bers made the pathological diagnosis of nemaline myopathy (Fig. 2a, b). Interesting is that the most of fi bers looked similar to those seen in cap myopathy, especially on ATPase staining (Fig. 2c. One month after the biopsy, before any genetic confi rmation could be obtained, the boy died of severe respiratory insuffi ciency and pneumonia.

Reducing body myopathy
There was only one patient in this group, a woman aged 25 years with late onset of symptoms. She had predominanly proximal assymetric scapuloperoneal weakness and scoliosis. CK level was three times the normal value and EMG revealed mixed neurogenic and myopathic changes. The biopsy performed in the right gastrocnemius muscle identifi ed a marked variation in the fi ber size, some necrotic fi bers, increased number of internally located nuclei, splitting of fi bers, small groups of atrophic fi bers, but the most prominent morphological aspect was the presence of granular bodies located in the cytoplasm, often near the nucleus, looking dark-green on modifi ed Gomori trichrome stain (Fig. 3). These aggregates reduce nitro-bluetetrazolium (NBT) and thus stain strongly with the menadione-NBT stain, presumably because of their high content of sulfhydryl group (Fig. 3). Under the high suspicion of reducing body myopathy, the case has been sent to Prof. M. Fardeau (Institute of Myology in Paris) for second oppinion and electron microscopy. The typical electron-dense granular fi la mentous appearance of the aggregates confi rmed our diagnosis.

Centronuclear/myotubular myopathies
We examined a total of fi ve cases with a significant number of centrally located myonuclei, as the hallmark of centronuclear myopathies (7). In two pediatric cases aged 9 and 7 years the single central nuclei were prominent and occupyed a large volume of the fi ber, highly suggesting centronuclear myopathy (Fig. 4). Other morphological features were reduced average fi ber size and predominance of type I fi bers that were smaller than type II fi bers. The central areas of the fi bers showed reduced myofi brillar ATPase reaction and decreased oxidative enzyme activity (Fig. 4). Another obvious features were myofi brils with radiating structure on oxidative enzyme stains looking like spokes of a wheel (Fig. 4). In both cases, the onset was in early childhood with severe hypotonia, proximal and dis-

FIGURE 4. Centronuclear myopathies: characteristic facial weakness, long face and ptosis (a); biopsy with morphological aspects of centronuclear myopathy: centrally located myonuclei mostly in small fi bers (b,c) (HE, GT); radial strands like spoke of wheel on NADH stain (d, e) and LDH stain (f) in some fi bers, and pale central areas devoid of mitochondria in other muscle fi bers (Original magnifi cation Ob. 40x), lacking cytochrome C oxidase activity (COX stain, Original magnifi cation Ob. 20x) (g)
tal simmetric nonprogressive weakness, ptosis and ophtalmoplegia, respiratory inssufi ciency and craniofacial dysmorphism consisting in a long face, dolichocephalic skull and high arched palate. The pronounced facial weakness, particularly affecting the lower face and mouth resulted in the characteristic "myopathic facies" (Fig. 4a). CK levels were mildly elevated and EMG showed myopathic features. The other three case were adults and among them there was a man aged 46 years with the clini-cal suspicion of muscular dystrophy. He had moderate and very slowly progressive weakness since early childhood predominantly in the proximal musculature, he was always poor in sport and had diffi culties walking and climbing stairs. CK levels were moderately elevated. His biopsy from the left deltoid muscle showed moderate variation in the fi ber size and a large number of internal nuclei. On oxidative enzyme stains we noticed a large number of fi bers with radiating strands of sarcoplasm from the central nucleus, but also other fi bers with corelike structures. The semithin sections showed, besides muscle fi bers with one or more central nuclei, many fi bers with central cores structures, unstructured sarcomeric fi brillar aggregates areas with prominent Z bands material, clear zones without myofi laments, a central accumulation of abnormal mithocondria (Fig. 5, a-j). The other two cases were females aged 22 and 36 years and also presented with proximal weakness. In all of the adult cases we noticed increased number of internally placed nuclei, moderate fi ber size variation and mild increase in endomysial connective tissue. Because of the large number of internal nuclei a differential diagnosis with myotonic dystrophy based on clinical signs and genetic tests is required in such cases.

Congenital fi ber type disproportion
During the study period we diagnosed three cases of congenital fi ber type disproportion (CFTD), all of them in children, aged 1 year and 9 months, 3 years and 12 years. All of the patients were males. While the younger child was very hypotonic and had severe respiratory insuffi ciency, the other two only presented mild diffuse weakness, reduced tendon refl exes and scoliosis, highlighting the wide variation of symptoms in CFTD. CK levels were normal in all of the cases. The muscle biopsies showed in all the three cases the characteristic bimodal distribution of fi ber sizes with type I fi bers being more numerous and smaller than type II fibers with at least 12% as the only morphological abnormality (Fig. 6).

DISCUSSIONS
CM are clinically, genetically but also morphologically very heterogenous diseases. For most of the frequent forms and even for some of the rarer ones the causative gene defects were identifi ed in the last years, revealing an interesting and complex spectrum. For each of the gene there is a wide phenotypic variation refl ected in very different age of onset from birth, with neonates who are not able to survive, while in other cases the disease starts in childhood or even in late adulthood, so that in most of the CM there are three distinct clinical subtypes: a severe infantile form, a benign congenital one and adult onset form. Moreover, the diversity of clinical pictures is completed by large overlap of pathology in CM, varying morphology being associated with defects in the same gene, while very similar pathology may be caused by defects in different genes (2). For these reasons, directing molecular analysis to reach a precise diagnosis requires a combination of clinical investigations and morphological assessment of frozen sections of muscle using routine, Gomori trichrome and oxidative enzyme stains and electronmicroscopy. Thus, is obvious that the muscle biopsy should be performed and analysed in specialised centres with facilities to work-up such cases. A piece of formalin-fi xed muscle tissue later embedded in paraffi n would certainly miss the diagnosis of CM.
In this study we were particularly interested in the pathological overlaps, more and more described in CM (8,9), because they may represent overlapping pathomechanisms. In all the analyzed cases we identifi ed pronounced type I predominance or even uniformity of fi ber types with type I hypotrophy. Another common morphological aspect is the increased number of internally located nuclei, and even the presence of a small number of muscle fibers with central core-like areas, not reactive on oxidative enzyme stains, suggesting lack of mitochondria COX activity. In our actual knowledge, cores and rods does not necessarily imply different clinical entities, but are part of a spectrum of morphological aspects (10,11). In the adult case of centronuclear myopathy presented above, we found on the same biopsy myonuclei in the center of many fi bers and cores in other fi bers, once again underlining the pathological overlapping. This pathological pattern was found in CM in adult patients with a BIN1 mutation (12), the genetic analysis proving to be very imprtant for a more accurate diagnosis. The variability of pathology appears to be related to the sampled muscle, the age at biopsy and to the change of morphology in time. Another extremely important practical aspect is that core myopathies with mutation in the ryanodine receptor gene carry a very high risk of malignant hyperthermia in response to anesthetic triggering agents, so that a precise diagnosis in such cases will help preventing this life-threatening complication (13)(14)(15)(16)(17). Another overlapping aspect we should mention is that of the pacient with nemaline myopathy. Many fi bers looked like those in "cap" myopathy, especially on APT-ase staining. Cap myopathy fi rst described in 1981 by Fidzianska and co-workers (18), was shown to be produced by a mutation in b-tropomyosin (TPM2) gene in a patient (19). The diversity of clinical phenotypes in CM is very convincingly refl ected in our studied cases. We diagnosed both young children with very severe weakness and re-spiratory insuffi ciency starting in the neonatal period, older children with milder symptoms but also adults with long history of their disease. A feature of our study is that we diagnosed many cases of CM in adulthood, suggesting that in our country CM are recognized late in the disease progression and are probably underdiagnosed.
The clinical differential diagnosis in CM is complex and include forms of congenital muscular dystrophies, congenital myotonic dystrophy, spinal muscular atrophy, congenital myasthenic syndromes, metabolic myopathies including Pompe disease, as well as Prader-Willi syndrome. Not surprisingly, some of these diagnosis were clinically suspected in our patients. Patterns of clinical fi ndings very highly suggestive for CM are prominent facial weakness with or without ptosis, generalised hypotonic posture, hyporefl exia, weakness and dysfunction of the respiratory and bulbar muscles. We could fi nd combinations of these symptoms in the clinical pictures of our patients.
We were also interested in comparing our retrospective study with others in terms of incidence of CM and of the different subtypes. We diagnozed CM in 15 cases, representing 1.03% of all the muscle biopsies performed in the time of our study and the most important types of CM were found in the Romanian population in the last 10 years. To compare, in the same period of time we examined 78 cases of Duchenne muscular dystrophy and 31 cases of Becker muscular dystrophy or intermediate Duchenne-Becker forms. Core myopathies and centronuclear myopathies had a equal frequency. In 2013 Maggi et al. published a study on 66 patients diagnosed with CM over a period of 5 years in the U.K, of which 54 had a muscle biopsy and concluded that core myopathies were the most common form of CM (20). Other studies from large countries like India indicated that 50 out of 3,420 muscle biopsies over a period of 12 years were diagnosed as CM, representing a percentage of 1.46%. Among the subtypes, the most prevalent type were centronuclear myopathies (21). In the large study of Nonaka out of 446 cases diagnosed in 21 years (in Japanese population), nemaline myopathy was the leading form of CM and the author concluded that CM are relatively common among the childhood myopathies (22). Thaha et al. in a study on 40 patients published in 2011 reported that centronuclear myopathy was the commonest type of CM, followed by congenital fi ber type disproportion (23). Jain et al retrieved all the cases diagnosed during a period of 6 years, and reported that 1.12% of the muscle biopsies were diagnosed as CM and the most common of them was central core disease followed by nemaline rod myopathy and multimini core disease (24). Obviously, there are differences between populations in the overall incidence and among subtypes. However, the global incidence of CM and of individual forms is yet mostly unknown,emphasizing the need to continue and improve their detection and this goal will be achieved through the widespread introduction of molecular genetic techniques, particularly next generation sequencing (25,26).
A precise diagnosis in CM is essential not only for prognostic purposes and early management of complications, but also for genetic counseling, in waiting for future specifi c genetic therapies. A key aspect is also the psychological impact of a clearly established diagnosis for the patient and his family.

CONCLUSIONS
The most common subtypes of CM: core myopathies, nemaline myopathy and centronuclear/ myotubular myopathy were all diagnosed in the Romanian population in the last 10 years. We diag-nosed children as well as adults, mild cases but also very severe ones. The unexpectedly high proportion of adult cases in our study suggests that in our country these diseases are often diagnosed late or even remain undiagnosed. Even now, in the molecular era, achieving a precise diagnosis requires correlations of the morphological aspects provided by the muscle biopsy using histological, histochemical and electron microscopy techniques with the clinical phenotype. The diffi culties in the diagnostic algorithm is increased by the clinical, genetical and pathological overlaps and demand a multidisciplinary approach of the cases.