The diagnostic value of vacuolar characterization in muscular biopsy in Pompe's Disease

Introduction. Through muscle biopsy we can observe the formation of vacuoles that alter the structure of cells and tissues in Pompe's disease. The presence of these vacuoles varies as the disease progresses, relating to the broad clinical spectrum presented by the disease. Objectives. After confirming the disease, examine the possibility of diagnosing or excluding the diagnosis of Pompe's disease through the vacuolar characteristics presented. Method. Analysis of the muscle biopsy material of selected patients at the Neuromuscular Diseases Investigation Clinic at the Federal University of São Paulo. Through staining and histochemical techniques, a comparative study of the histological characteristics found was performed. Results. Thirty-three biopsies had the diagnosis of Pompe's disease confirmed, being 13 women and 20 men. Of this group, 23 received the diagnosis when they were 18 years old or more, and 10 received the diagnosis under the age of 18 years. Delimiting membrane and subsarcolemal location were the main vacuolar characteristic found, manifesting in 86.6% of the studied cases. Integration between the vacuole membranes was observed in 62.5% of the

cases. We also found necrosis, replacement of muscle tissue by connective or adipose tissue, increased mitochondrial activity and absence of predominance in one type of fiber. Conclusion. Muscle biopsy allows to analyze a series of peculiarities presented by vacuoles in Pompe's Disease and, thus, it proves to be a sure technique, allowing to reach a quick conclusion and to identify determining factors for the clinical conduct and maintenance of quality of life of the patient with Pompe's disease. Keywords. Pompe disease; Neuromuscular disease; Biopsy; Histology; Cytopathology Resumen Introducción. Mediante biopsia muscular podemos observar la formación de vacuolas que alteran la estructura de células y tejidos en la Enfermedad de Pompe. La presencia de estas vacuolas varía a medida que avanza la enfermedad, en relación con el amplio espectro clínico que presenta la enfermedad. Objetivos. Una vez confirmada la enfermedad, examinar la posibilidad de diagnosticar o excluir el diagnóstico de la enfermedad de Pompe a través de las características vacuolares presentadas. Método. Análisis del material de biopsia muscular de pacientes seleccionados en la Clínica de Investigación de Enfermedades Neuromusculares de la Universidad Federal de São Paulo. Mediante técnicas de tinción e histoquímica se realizó un estudio comparativo de las características histológicas encontradas. Resultados. En 33 biopsias se confirmó el diagnóstico de enfermedad de Pompe, siendo 13 mujeres y 20 hombres. De este grupo, 23 recibieron el diagnóstico cuando tenían 18 años o más, y 10 recibieron el diagnóstico antes de los 18 años. La membrana límite y la localización subarcolémica fue la principal característica vacuolar encontrada, manifestándose en el 86,6% de los casos estudiados. La integración entre las membranas de las vacuolas se observó en el 62,5% de los casos. También encontramos necrosis, sustitución de tejido muscular por tejido conectivo o adiposo, aumento de la actividad mitocondrial y ausencia de predominio en un tipo de fibra. Conclusión. La biopsia muscular permite analizar una serie de peculiaridades que presentan las vacuolas en la Enfermedad de Pompe y, así, resulta ser una técnica segura, permitiendo llegar a una conclusión rápida e identificar factores determinantes para la conducta clínica y el mantenimiento de la calidad del paciente, de la vida con la enfermedad de Pompe. Palabras clave. Enfermedad de Pompe; Enfermedades neuromusculares; Biopsia; Histología; Citopatología

INTRODUCTION
Within the so-called group of inborn errors of metabolism, we can find the glycogen storage disease type II or Pompe's disease 1 . This can be defined generically as a neuromuscular, genetic, metabolic, and rare disease, which leads to low or complete absence of the production of an enzyme. This enzyme deficiency causes the accumulation of glycogen inside the cells, especially in the muscles and liver, configuring the symptoms muscle weakness, fatigue, and pain, and can manifest at any age 2 . This accumulation is caused by a deficiency in the activity of the acid alpha-glucosidase lysosomal enzyme (GAA), which catalyzes the breakdown of glycogen into glucose; at low pH (4.0-5.0), through the hydrolysis of the portions to alpha-1. 4  On the other hand, the quantification of the activity of the GAA enzyme by techniques such as dried blood stain has been gaining ground due to its precision and speed, in addition to being a non-invasive and safe method for the patient 9 . However, tests like this are only applied in cases of strong suspicion, and often with a long clinical history, and its execution is limited to some laboratories 10 .
Considered as a complementary test or an alternative for enzyme dosage tests and molecular tests, a muscle or skin biopsy is an important option, as it is possible to identify a disease, in some cases, through specific histopathological findings 11 .  Both characteristics, delimiting membrane and subsarcolemal location, could be observed in 86.6% of the studied cases. The type of vacuole most commonly observed is the rimmed vacuole type, which is an important characteristic as a classificatory factor. An important characteristic for the definition of the disease would be the quantity of vacuoles, as well as its size, however these characteristics proved to be difficult to define because they vary according to the degree of patient affection and his age.
In glycogenesis, regardless of the form of the disease, as it progresses, there is an increase in the accumulation of glycogen within the lysosomes, which expand and may even merge with each other, giving rise to vacuoles whose length can occupy more than half the size of the fiber muscle, generating change in the internal structure, which reflects clinically with decreased muscle strength, decreased mobility and, consequently, progressive loss of muscle function. In our population, we found 62.5% of cases with fuse between the vacuole membranes, of which 81.8% were diagnosed with the late form.
We found necrosis in 25% of cases. Another memorable finding, unlike inflammatory diseases, is the infrequent encounter of an inflammatory process in glycogenoses, however with the replacement of muscle tissue by connective or adipose tissue in 81.25% of the analyzed cases.
Although studies suggest that there is no predilection for the formation of vacuoles in a specific type of muscle fiber, defining whether there is a predominance of one type is important for enzyme replacement therapy because recent studies indicate differences in the numbers of mannose-6phosphate receptors between the types of fibers. Research has also shown that type II fibers are more resistant to TRE 14 .
Usually, there are no signs of muscle fiber atrophy, change on the fiber diameter or predilection for some type of muscle fiber in glycogenoses. In cases with Pompe's Disease, we found two cases with type II atrophy and one case with predominance of type II fibers.   Even with the absence of these techniques, we analyzed several muscle biopsies and were able to observe that certain vacuolar characteristics, which alongside other findings, strongly favor the diagnosis of this disease.