Essential neuromuscular advice for pathologists (first of two parts)

Abstract

Background

Neuromuscular disorders are characterized by disturbances in any part of the neurologic pathways, including: the Central Nervous System, the motor neuron of the anterior horn of the spinal cord; the peripheral nerve, the neuromuscular junction, and the muscle. Neuromuscular disorders are considered rare affections but when the prevalences of all subtypes are analysed together they may be encountered by general neurologists and pathologists. Therefore, basic knowledge in this field is necessary to timely guide serologic, molecular, or muscle biopsy investigation for appropriate treatment and/or genetic counselling.

Main body

The aims of this review are: (1) to briefly describe the prevalence of common neuromuscular disorders; (2) to present basic concepts of topographic neuromuscular diagnosis; (3) to provide essential information for pathologists about the diagnostic approach of common neuromuscular disorders; (4) to present basic concepts of muscle imaging for myopathologists; (5) to provide muscle imaging, and biopsy examples of common neuromuscular disorders.

Conclusion

A multiprofessional integrated approach is essential for precise neuromuscular diagnosis. Detailed clinical examination with the formulation of phenotypic hypothesis is the basis for appropriate diagnosis in the Surgical-Molecular Pathology era. Clinical, epidemiological, neurophysiological, laboratorial, imaging, molecular, and physiopathologic aspects are essential for adequate neuromuscular diagnosis.

Background

Neuromuscular disorders are considered rare affections but when the prevalences of all subtypes are analysed together they may be encountered by general neurologists and pathologists. Therefore, basic knowledge in this field is necessary to timely guide serologic, molecular, or muscle biopsy investigation for appropriate treatment and/or genetic counselling.

Myopathies are muscular disorders that affect the normal function of the muscle. They may be acquired, such as toxic myopathies or autoimmune myositis, or they may be hereditary. Limb Girdle Muscular Dystrophy is defined as a group of progressive autosomal recessive (85%) and autosomal dominant (15%) muscular dystrophies described in at least two unrelated families, affecting individuals that achieve independent walking, with predominant proximal muscles weakness at presentation, with elevated serum creatine kinase activity, dystrophic changes on muscle biopsy (Fig. 1), and degeneration on muscle imaging over the course of the disease.

Fig. 1

Muscle biopsy architectural characteristics in normal (a, b, and c) and dystrophic muscle (d, e, f, g, h, i, j, k, and l). Normal muscle biopsy from a 6 years old male patient suspected of congenital myopathy submitted to right vastus lateralis muscle biopsy that presented normal general architecture with very mild variation the same calibre with mild connective tissue in the perimisium (yellow arrows in a, b, and c) separating muscle fascicles, and almost imperceptible endomysium (white arrows in b and c) between muscle fibres that present nuclei (green arrows in b) disposed at the periphery of the fibres. Muscle biopsy with dystrophic pattern from a 22 years old male patient submitted to right tibialis anterior muscle biopsy for immunohistochemical confirmation of the pathogenicity of a homozygous VUS (variant of unknown significance) c.3235T > C p.(Cys1079Arg), in the exon 23 of the LAMA2 gene. The dystrophic pattern is characterized by abnormal architecture with endomysial fibrosis (white arrow in d) that results in fibrous replacement of the muscle tissue followed by muscle fat replacement (brown arrows in e, f, and i) with striking variation in fibre calibre with hypertrophy (yellow asterisks * in d, e, and j), atrophy (white asterisks * in d, e, f, and j); hypertrophic fibres may present fibre splitting (yellow arrow in d) and abnormally internalized nuclei (green arrow in d); the driver physiopathological events that result in the dystrophic pattern are necrosis (pale pink area of coagulative necrosis pointed by the white arrow in g, and pale green area pointed by the yellow arrow in k), phagocytosis (macrophages with abundant cytoplasm pointed by the yellow arrow in f, and g), and regeneration (muscle fibre with weak basophilic sarcoplasm and increased internalized nucleus pointed by the yellow arrow in h). Pathogenicity confirmation of the VUS in LAMA2 gene was demonstrated by irregular laminin-alpha2 imunohistochemical reaction (blue arrow in l), compared to the normal control (red arrow in the inset in l) (a. HE 100x, b. HE 200x, c. Modified Gomori trichrome 200x, d. HE 100x, e. HE 100x, f. HE 100x, g. HE 200x, h. HE 200x, i. Modified Gomori trichrome 25x, j. Modified Gomori trichrome 100x, k. Modified Gomori trichrome 200x, l. Immunohistochemistry anti-laminin-alpha-2 (anti-merosin) 200x, patient and control inset). VUS: variant of unknown significance)

The main goals of this review are: (1) to briefly describe the prevalence of common neuromuscular disorders; (2) to present basic concepts of topographic neuromuscular diagnosis; (3) to provide essential information for pathologists about the diagnostic approach of common neuromuscular disorders; (4) to present basic concepts of muscle imaging for myopathologists; (5) to provide muscle imaging and biopsy examples of common neuromuscular diagnosis.

It is beyond the scope of this review to provide a detailed description of all myopathies. For complementary information, please refer to classic myopathology textbooks and reviews (Engel and Franzini-Armstrong 2004; Karpati et al. 2010; Dubowitz et al. 2020; Dubowitz et al. 2013; Dubowitz and Sewry 2007; Dubowiz 1995; Dubowitz and Brooke 1973; Loughlin 1993; Anderson 1985; Amato and Russell 2008; Levy 1978; Dastgir et al. 2016, Fardeau 2017; Malfatti and Romero 2017; Nix and Moore 2020, and Udd et al. 2019).

Prevalence of neuromuscular disorders

In this section we present a brief overview of the literature on the prevalence (number of cases in a population regardless of the time of the first symptoms) of neuromuscular disorders that may be found frequently in a rehabilitation centre (Table 1) (Amatangelo et al., 2020; Balestrassi et al., 2021; Benarroch et al. 2024; Cavalcanti et al. 2020; Deenen et al. 2015; Finsterer 2019; Finsterer et al. 2021; GBD 2020; GBD 2021; Gentile et al. 2022; Gorman et al. 2015; Gusic and Prokisch 2021; Hoytema van Konijnenburg et al. 2021; Long et al. 2022; Mihaylova et al. 2010; Mortier et al. 2019; Norwood et al. 2009; Parr et al. 2014; Pringsheim et al. 2014; Ruano et al. 2014; Singh et al. 2015; Svensson et al. 2017; Thompson et al. 2019; Uchoa Cavalcanti et al., 2021; Winckler et al. 2019). In order to provide general epidemiological context for pathologists, the incidence (number of new cases in a time period) of the most common cancers in Brazil is presented in Table 2 (Instituto Nacional de Câncer/ Brasil (2022)).

Table 1 Prevalence of selected clinical disorders in neuromuscular practice (per 100,000 inhabitants) and current management (Amatangelo et al., 2020; Balestrassi & Silva (2021); Benarroch et al. 2024; Cavalcanti et al. 2020; Deenen et al. 2015; Finsterer 2019; Finsterer et al. 2021; GBD 2020; GBD 2021; Gentile et al. 2022; Gorman et al. 2015; Gusic and Prokisch 2021; Hoytema van Konijnenburg et al. 2021; Long et al. 2022; Mihaylova et al. 2010; Mortier et al. 2019; Norwood et al. 2009; Parr et al. 2014; Pringsheim et al. 2014; Ruano et al. 2014; Singh et al. 2015; Svensson et al. 2017; Thompson et al. 2019; Uchoa Cavalcanti et al., 2021; Winckler et al. 2019)

Table 2 Estimated cancer adjusted incidence in Brazil (per 100,000 inhabitants), except melanoma and skin cancer (INCA 2022)

Among neuromuscular disorders, as a general rule, neuropathies are more frequent than myopathies and acquired (potentially treatable) conditions are more prevalent than hereditary diseases. In children, secondary hypotonia is caused by several conditions such as hypoglycemia, congenital cardiopathy, and sepsis, while primary hypotonia with muscle weakness may be related to central causes (60%-80%) or to peripheral causes such as neuromuscular disorders (15%-30%) including myopathies, and congenital myasthenic syndromes (Morton et al. 2022).

Basic concepts of topographic neuromuscular diagnosis

Neuromuscular disorders are characterized by disturbances in any part of the neurologic pathways, including: the Central Nervous System (e.g. babies with central hypotonia and structural lesions in the brain); the motor neuron of the anterior horn of the spinal cord (e.g. Spinal Muscular Atrophy; Poliomyelitis, etc.); the peripheral nerves (e.g. diabetic neuropathy); the neuromuscular junction (e.g. Myasthenia gravis, Congenital Myasthenic Syndromes); the muscle (e.g. dermatomyositis; Duchenne muscular dystrophy, etc.). Myopathies are characterized by a primary affection of the muscles (Fig. 2) (Guyton and Hall 2006; Drake et al. 2021).

Fig. 2

Topographic localization of neuromuscular disorders (Guyton and Hall 2006; Drake et al. 2021). a The first motor stimulus starts in the Primary Motor Neuron in the Primary Motor Cortex in the Precentral gyrus and follows through the Pyramidal Tract. It passes through the posterior limb of internal capsule, genu of corpus callosum, basis pedunculi of mesencephalon, longitudinal fascicles of pons, pyramid of medulla oblongata, ventral corticospinal tract (data not shown) until the Lower Motor Neuron in the Anterior Horn of the Spinal Cord. b The Lower Motor Neuron (blue) connects through the axon (blue) to the Neuromuscular Junction (NJM) (green), and to the muscle (red). c Neuromuscular Junction (NMJ) with Schwann cell, axon terminal, NMJ post-synaptic folds, and muscle. Transmission Electron Microscopy (6,000x) (c) of a 43 years old female patient with congenital myasthenic syndrome with simplification of the post-synaptic folds

Some clinical signs may be helpful to the differential diagnosis between myopathies and other neuromuscular affections with proximal weakness such as normal or decreased deep tendon reflexes in myopathies, fasciculations in motor neuron diseases, and sensory loss in peripheral neuropathies (Table 3) (Bertorini 2022). The patient with myopathy may present either positive signs, such as muscle hypertrophy and myalgia, or negative signs such as muscle atrophy and weakness (Table 4) (Bertorini 2022; Jackson and Bahron 2013; Pasnoor and Dimachkie 2019). The clinical symptoms and signs may provide clues to the pattern of weakness that may be correlated to the respective muscles affected (Table 5) (Bertorini 2022; Gerth and Festman 2023; Nicolle 2016; Pasnoor and Dimachkie 2019).

Table 3 Neurologic topographic localization and respective clinical signs (modified from Bertorini 2022)

Table 4 Basic neurological concepts for the clinical evaluation of patients with myopathies (Bertorini 2022; Jackson and Bahron 2013; Pasnoor and Dimachkie 2019)

Table 5 Symptoms, signs and respective affected muscles in myopathies (Bertorini 2022; Gerth and Festman 2023; Nicolle 2016; Pasnoor and Dimachkie 2019)

Clinical-molecular-pathological diagnostic approach to the patient with myopathy

Muscular Dystrophies are clinically characterized by muscle weakness. Each muscular dystrophy presents a general pattern of muscle weakness that may be used for differential diagnosis (Fig. 3) (Emery 1998; Emery 2002a; Emery 2002b; and Wenninger et al. 2018). The general muscle weakness pattern may be suspected by the main complains at the anamnesis and they may be confirmed by neurological physical examination.

Fig. 3

Clinical differential diagnosis of myopathies based on weakness pattern (modified from Emery 1998; Emery 2002a; Emery 2002b; and Wenninger et al. 2018). Weak muscle groups are represented in blue. a Patients with Limb Girdle Muscular Dystrophy (LGMD) present proximal upper limbs, proximal lower limbs, shoulder-girdle, and pelvic-girdle weakness. b Patients with Dystrophinopathy included in the spectrum from the most severe Duchenne Muscular Dystrophy to the milder Becker Muscular Dystrophy present proximal upper limbs, proximal lower limbs, and limb girdle weakness combined with prominent increased volume (not shown), and weakness of the calf muscles (pseudohypertrophy). c Patients with Proximal Myotonic Myopathy (PROMM or Myotonic Dystrophy type 2) present weakness that is more prominent in the pelvic girdle (hip flexors and hip extensors), and in the neck flexors, followed by proximal upper limbs, and limb girdles. d Patients with Myotonic Dystrophy type 1 (Steinert´s myotonic dystrophy) present distal muscular atrophy and weakness mainly involving wrist flexors, finger flexors, and foot extensors, combined with a characteristic myopathic facial phenotype with forehead balding, temporal wasting, and ptosis combined with nasal speech, and dysphagia. e Patients with Facioscapulohumeral Muscular Dystrophy (FSHD) present characteristic muscle weakness that involves the scapular girdle with limitation of arm abduction, and scapula alata (not shown), combined with leg weakness, and facial weakness with difficulties to close the eyes, to protrude the lips, and to puff out the cheeks. f Emery-Dreifuss muscular dystrophy starts with early joint contractures (not shown) of the elbows, Achilles tendons, and posterior cervical muscles that progresses to a humeroperoneal pattern of muscle weakness that involves the scapular girdle and leg muscles followed by cardiomyopathy with cardiac conduction defects (not shown). g Oculopharyngeal muscular dystrophy (OPMD) presents involvement of the extraocular muscles, upper facial muscles with ptosis, neck musculature, proximal upper limbs, and lower limbs muscles. h Distal muscular dystrophies (previously known as distal myopathies) present weakness of distal upper limbs, and distal lower limbs muscles

A thorough clinical and neurologic investigation with a complete anamnesis including familial history and detailed neurologic examination provide the basis for a precise diagnosis. The clinical hypotheses guide specific ancillary exams to confirm the diagnosis. The electroneuromyogram (EMG) is an extension of the neurological examination and, in experienced hands, it may discriminate between myopathic and neurogenic disorders (Hafner et al. 2019). The diagnostic yield of EMG may be as high as 88% for myopathic processes even when serum muscle enzymes are normal (Cotta et al. 2022). Besides this, electroneuromyogram with repetitive nerve stimulation may provide the phenotypic confirmation of neuromuscular junction defects, that are of utmost importance in the growing group of treatable Congenital Myasthenic Syndromes (Table 1) (Finsterer 2019; Thompson et al. 2019).

With the advent of new technologies, such as Next Generation Sequencing (NGS), molecular investigation has become the second step in the investigation of myopathies, after the elaboration of precise clinical diagnostic hypothesis (Fig. 4) (Cotta et al. 2017, 2019; Nicolau et al. 2021; Nishikawa et al. 2017; Straub et al. 2018; Verdu-Diaz et al. 2020).

Fig. 4

Algorithm for the differential diagnosis of myopathies (Cotta et al. 2017, 2019; Nicolau et al. 2021; Nishikawa et al. 2017; Straub et al. 2018; Verdu-Diaz et al. 2020). The first step in the diagnosis of hereditary myopathies is to exclude the more common and potentially treatable acquired disorders with clinical evaluation and ancillary tests. CPK: creatine phosphokinase; EMG: electromyogram; electroneuromyogram; MLPA: Multiplex Ligation-dependent Probe Amplification; PCR: polymerase chain reaction; RNS: repetitive nerve stimulation; STIR: Short Tau Inversion Recovery sequence on magnetic resonance imaging; T1: T1-weighted longitudinal relaxation time sequence on magnetic resonance imaging; TSH: thyroid stimulating hormone

There has been a profound transformation in neuromuscular practice and diagnostic investigation. Pathologists should be aware of the various modalities of molecular exams to choose the appropriate technique for each patient according to the disease mechanism (da Cunha et al. 2021). Myopathies related to single base pair pathogenic variants may be diagnosed by Next Generation Sequencing (NGS); other conditions need different molecular diagnostic techniques (da Cunha et al. 2021) such as Multiplex Ligation-dependent Probe Amplification (MLPA) for large deletions and duplications (Araujo et al. 2018). Figure 4 presents a general algorithm for the differential diagnosis of the most common myopathies (Cotta et al. 2017, 2019; Nicolau et al. 2021; Nishikawa et al. 2017; Straub et al. 2018; Verdu-Diaz et al. 2020). In the first step of diagnosis, a complete neurological evaluation may be performed focused mainly on the determination of the clinical hypothesis exclusion of potentially treatable diseases, and the most common inherited neuromuscular diseases.

Basic concepts of muscle imaging for pathologists

Muscle imaging has increasingly become a large part of neuromuscular practice during the last decades. Excellent pioneer works on muscle ultrasound imaging in children with muscular dystrophy have been published since the 1980s (Heckmatt et al. 1980, 1982). Recently, muscle Magnetic Resonance Imaging (MRI) (Warman Chardon et al. 2019; Venturelli et al. 2023), and muscle ultrasound (Albayda and van Alfen 2020) have progressively increased their role in the initial evaluation of neuromuscular patients to guide proper diagnosis (Albayda and van Alfen 2020). Whole-body magnetic resonance imaging allows the detection of characteristic radiophenotypes of several myopathies and it has become the imaging modality of choice by the MYO-MRI Working Group sponsored by the World Muscle Society (Warman Chardon et al. 2019). Some limitations have been found to implement whole-body MRI in routine diagnosis outside research protocols. Therefore, in this review, we present data based on our experience with the routine use or axial T1-weighted and STIR-weighted Magnetic Resonance Images of the pelvis, thighs, and legs that have been used in the initial evaluation of all patient with myopathies admitted in our service. It is well known that brain imaging has been used in routine neuropathology practice for the diagnosis of brain tumors. Likewise, muscle imaging has been used as a surrogate of "gross examination" for muscle biopsies.

Here we present basic anatomic concepts useful for the evaluation of axial T1-weighted images of the pelvis, thighs, and legs in normal muscle (Fig. 5) (Drake et al. 2021). A comparative analysis of different imaging modalities with muscle grading scales is presented in Fig. 6 (Wattjes et al. 2010; Heckmatt et al. 1982; Cotta et al. 2014, and personal files).

Fig. 5

Normal Muscle imaging in the evaluation of myopathies (modified from Drake et al. 2021, and personal files). a planes for examination: coronal plane is a vertical plane (green polygon in a, and b) that divides the body in anterior and posterior sections, sagittal plane is a vertical plane (blue polygon in a, and c) that separates the body in right and left halves, and transverse or axial plane (red polygon in a, and d, and blue lines in e) that is a horizontal plane, parallel to the ground, perpendicular to both the sagittal and coronal planes. Muscle diagram (f, g, and h) and Axial T1-weighted magnetic resonance imaging (i, j, k) of the pelvis (f and i), thighs (g and j), and legs (h and k) from a 10 years old male patient without muscular dystrophy. The thigh (g) and legs (h) are divided in anterior (green area in g and h), medial (blue area in g), posterior (orange area in g, and h), and lateral (red area in h) compartments. Muscles of the pelvis (f and i): MA: Gluteus maximus; ME: Gluteus medius; and MI: Gluteus minimus. Muscles of the thighs (g and j): VL: Vastus lateralis; VM: Vastus medialis; VI: Vastus intermedius; RF: Rectus femoris; S: Sartorius; G: Gracilis; AM: Adductor magnus; AL: Adductor longus; BF: Biceps femoris long head; BS: Biceps femoris short head; ST: Semitendinosus; and SM: Semimembranosus. Muscles of the legs (h and k): TA: Tibialis anterior; E: Extensor group (extensor digitorum longus, and extensor hallucis longus); P: Peroneus group (peroneus longus, and peroneus brevis); TP: Tibialis posterior; SO: Soleus; GM: Gastrocnemius medialis; and GL: Gastrocnemius lateralis. R: right, L: left

Fig. 6

Muscle imaging grading (Wattjes et al. 2010; Heckmatt et al. 1982; Cotta et al. 2014, and personal files). Each line shows the comparative muscle grading and each column represents different imaging modalities: MRI (magnetic resonance imaging) (a, d, g, j, and m), CT (computed tomography) (b, e, h, k, and n), and US (ultrasound) (c, f, i, l, and o). Mercuri scale is divided in grades 1 to 4; grade 1: normal appearance, 2: mild involvement of less than 30% of the volume of the individual muscle, 3: moderate involvement of 30–60% of the volume of the individual muscle, 4: severe involvement with washed out appearance. Fischer scale is divided in grades 0 to 4; grade 0: normal appearance; 1: mild traces of involvement; 2: moderate involvement of less than 50% of the muscle; 3: severe involvement of more than 50% of the muscle; 4: end-stage appearance. Ultrasound grading according to Heckmatt scale: grade (1): normal (c); (2): increased muscle echo while bone echo (blue arrow in f) is distinct; (3): marked increase in muscle echo, and reduced bone echo (i, and l); and (4): very strong muscle echo and complete loss of bone echo (o). Each line represents the same grade of muscle fat replacement: Mercuri 1/ Fischer 0/ Heckmatt (1) (arrows in a, b, and c); Mercuri 1/ Fischer 1/ Heckmatt (2) (arrows in d, e, and f); Mercuri 2/ Fischer 2/ Heckmatt (3) (arrows in g, h, and i); Mercuri 3/ Fischer 3/ Heckmatt (3) (arrows in j, k, and l); and Mercuri 4/ Fischer 4/ Heckmatt (4) (arrows in m, n, and o)

General guidelines for the diagnosis of the most prevalent neuromuscular disorders

A brief overview of the diagnostic criteria of the most prevalent neuromuscular disorders is illustrated in algorithms (Figs. 4, 7, 8, 9, 10, and 11), and molecularly proven cases with clinical-imaging-pathological correlation (Fig. 12).

Fig. 7

Myotonic dystrophy clinical and molecular diagnostic diagram (Catalli et al. 2010; Udd and Krahe 2012; Wenninger et al. 2018; Gutiérrez Gutiérrez et al. 2020; Hamel 2022). Muscle biopsy is not necessary for diagnostic confirmation of Steinert´s myotonic dystrophy type 1 and molecular exams may be ordered in the first clinical examination if the characteristic clinical features are detected. The diagnosis of myotonic dystrophy type 2 requires a high level of clinical suspicion to guide the correct molecular confirmatory tests. CCTG expansion: cytosine-cytosine-thymine-guanine tetranucleotide repeat expansion; CLCN1 gene: chloride voltage-gated channel 1 gene; CNBP gene: CCHC-type zinc finger nucleic acid binding protein gene; CTG expansion: cytosine-thymine-guanine trinucleotide repeat expansion; DMPK gene: dystrophia myotonica protein kinase gene; yo: years old; ZNF9 gene (now CNBP gene): zinc finger protein 9 gene

Fig. 8

Dystrophinopathy clinical and molecular diagnostic diagram (Araujo et al. 2018, 2023; Viggiano et al. 2023). Dystrophinopathy diagnosis is confirmed by molecular studies in about 95.8% of the patients and muscle biopsy is strictly necessary for diagnostic confirmation in only 4.2% of the patients. aCGH: array comparative genomic hybridization; ALT: alanine aminotransferase; AST: aspartate aminotransferase; CPK: creatine phosphokinase or creatine kinase; DMD gene: dystrophin gene or Duchenne muscular dystrophy gene; IPx: immunoperoxidase/ immunohistochemistry; MLPA: Multiplex Ligation-dependent Probe Amplification; NGS: next generation sequencing

Fig. 9

Facioscapulohumeral Muscular Dystrophy diagnostic diagram (Lamperti et al. 2010; Ricci et al. 2016; Mul 2022; Pasnoor and Dimachkie 2019). Facioscapulohumeral Muscular Dystrophy is clinically characterized by the triad of: facial weakness, scapular winging, and asymmetric limb muscle weakness. Facial weakness may manifest as transverse smile, sleeping with the eyes partially opened, inability to pucker, whistle, or drink from a straw. Palpebral ptosis and extraocular muscles are usually spared; this is important clue for the differential diagnosis with mitochondrial myopathies, as ophthalmoparesis is a cardinal feature of the Mitochondrial myopathy of the subtype Progressive External Ophthalmoplegia. Scapular winging is caused by weakness of the serratus anterior and trapezius muscles with inability to lift the arms, combined with wasting of the triceps brachialis, and biceps brachialis muscles, resulting in “Popeye” arms (reference to the cartoon character) with sparing of the forearm muscles. The diagnosis is confirmed by molecular studies and muscle biopsy is usually not performed, except in rare cases for differential diagnosis. D4Z4: macrosatellite (large tandem repeat) in the subtelomeric region of chromosome 4q35 (that contains the DUX4 gene); DUX4 gene: double homeobox 4 gene; FSHD1: Facioscapulohumeral Muscular Dystrophy type 1. Neurological physical examination grading by MRC (Medical Research Council): 1 Trace contraction of the muscle; 2 Ability to move with gravity eliminated; 3 Active movement against gravity; 4 Ability to move the joint against combination of gravity, and some resistance; and 5 Normal power

Fig. 10

Spinal muscular atrophy diagnostic diagram (Oskoui and Servais 2023; Arnold 2018). 5q-Spinal Muscular Atrophy is caused in 96% of the patients by a homozygous deletion of the SMN1 (Survival Motor Neuron) gene that causes a loss of function of the SMN protein. It is a common hereditary cause of muscle weakness and arreflexia with neurogenic electroneuromyography. The diagnosis is confirmed by molecular studies and muscle biopsy is not required for diagnosis. mRNA: messenger ribonucleic acid

Fig. 11

Oculopharyngeal muscular dystrophy, oculopharyngodistal myopathy and mitochondrial disorders diagnostic diagram (Yamashita et al., 2021; Stevanovski et al. 2022; Kumutpongpanich et al. 2021; Ogasawara et al. 2020, 2022; Gorman et al. 2015; Gusic and Prokisch 2021). Molecular studies confirm oculopharyngeal muscular dystrophy and oculopharyngeal muscular dystrophy diagnosis, therefore muscle biopsy is usually not necessary. Muscle tissue may be necessary for genetic studies of the mitochondrial DNA to avoid false negative results in DNA extracted from blood. Muscle biopsy may confirm the phenotype of mitochondriopathy with abnormalities in respiratory chain studies, histochemical proof of dysfunction with ragged red fibres (RRF), cytochrome-c-oxidative negative or COX (-) fibres, and ultrastructural abnormalities of the mitochondriae. bp: base pairs; COX(-): cytochrome c oxidase negative fibres; DNA: deoxyribonucleic acid; EEG: electroencephalogram. EM: electron microscopy. FGF-21: fibroblast growth factor 21; GDF-15: growth differentiation factor 15; GIPC1: GIPC PDZ domain containing family member 1 gene; IPx: immunohistochemistry; KSS: Kearns-Sayre syndrome; LHON: Leber hereditary optic neuropathy; LRP12: Low-density lipoprotein receptor-related protein 12 gene; MELAS: Mitochondrial myopathy, Encephalopathy, Lactic Acidosis, and Stroke-like episodes; MERRF: Myoclonic Epilepsy associated with Ragged-Red Fibres; MILS: maternally inherited Leigh Syndrome; mtDNA: mitochondrial deoxyribonucleic acid; NARP: neuropathy, ataxia, and retinitis pigmentosa; NOTCH2NLC: Notch homolog 2 N-terminal-like protein C gene; OPDM: oculopharyngodistal myopathy; OPMD: oculopharyngeal muscular dystrophy; PABPN1 gene (OPDM): polyadenilate-binding protein nuclear 1; POLG1 gene: polymerase, DNA, gamma-1 gene; RILPL1: PEO: progressive external ophthalmoplegia; Rab interacting lysosomal protein like 1 gene; yo: years old

Fig. 12

Muscle imaging and light microscopy findings of genetically proven common neuromuscular genetic diseases: Steinert´s type 1 myotonic dystrophy (a, b, c, d, and e), Dystrophinopathy (f, g, h, i, and j), Facioscapulohumeral Muscular Dystrophy (k, l, m, n, and o), Spinal Muscular Atrophy (p, q, r, s, and t), Oculopharyngeal Muscular Dystrophy (u, v, w, x, and y), and Mitochondriopathy (z, α, β, γ, and δ). Muscle MRI of the pelvis (a), thighs (b), and legs (c) and muscle biopsy (d and e) of a 54 years old female patient with Steinert´s myotonic dystrophy type 1 confirmed by the detection of a EcoRI restriction fragment larger than 10Kb correspondent to expansion of the CTG triplet in the DMPK gene with muscle fat replacement of the vastus intermedius (green asterisk * in b), soleus (yellow asterisk * in c), and gastrocnemius medialis (red asterisk * in c), nuclear internalization (black arrow in d), and ring fibres (black arrow and green asterisk* in e) with areas of myofibrillar loss and disorganization in subsarcolemmal crescents characteristic of subsarcolemmal sarcoplasmic masses (yellow arrow in d and yellow asterisk * in e). Muscle MRI of the pelvis (f), thighs (g), and legs (h) of a 28 years old male patient, and muscle biopsy (i and j) of a 16 years old male patient, both patients with Dystrophinopathy subtype Becker muscular dystrophy related to a deletion in exons 45 to 47 of the DMD gene. Muscle imaging demonstrates the trefoil with single fruit sign (illustration in g, drawn based on the figure created and published by Zheng et al. 2015) characterized by muscle fat replacement of the thigh muscles with hypertrophy of the semitendinosus (orange asterisk * and single fruit in g), and three leafs represented by sartorius (green asterisk * and green leaf in g), gracilis (red asterisk * and green leaf in g), and adductor longus (yellow asterisk * and green leaf in g); muscle biopsy with necrosis and phagocytosis (yellow arrow in i), and decreased intensity in the immunohistochemical reaction for dystrophin (black arrow in j) compared to the dystrophin intensity in the control (green arrow in the inset in j). Muscle MRI of the pelvis (k), thighs (l), and legs (m) of a 42 years old male patient and muscle biopsy (n and o) of a 58 years old female patient, both patients with Facioscapulohumeral Muscular Dystrophy molecularly confirmed by EcoRI/AvrII restriction fragments smaller than 35 Kb in the D4Z4 locus inside 4q35; muscle imaging shows asymmetric muscle fat replacement that is more severe in right adductor magnus (green asterisk * in l) compared to the left adductor magnus (white asterisk * in l), decreased right calf volume with severe muscle fat involvement of the right soleus (yellow asterisk * in m) compared to the preserved left side (white asterisk * in m), involvement of the right gastrocnemius medialis (red asterisk * in m) compared to the preserved left side (green asterisk * in m), bilateral foot drop (not shown) was related to severe muscle fat involvement of the right tibialis anterior (green arrow in m), and left tibialis anterior (yellow arrow in m); muscle biopsy with nuclear clumps (yellow arrows in n and o), nuclear internalization (white arrow in n), and atrophic fibres with increased acid phosphatase reaction (white arrow in o). Muscle MRI of the pelvis (p), thighs (q), and legs (r) and muscle biopsy (s and t) of a 16 years old male patient with molecularly confirmed Spinal Muscular Atrophy with homozygous deletion of the exons 7 and 8 of the SMN1 gene, with the SMN2 gene presenting 4 copies of the exon 7 and 3 copies of the exon 8; muscle imaging shows atrophy of vastus intermedius (yellow asterisk * in q), vastus medialis (green asterisk * in q) and sartorius (white asterisk *in q) that presents an imaging pattern of decreased volume and muscle involvement by adipose tissue that has been described in neurogenic conditions (Astrea et al. 2022) and are useful for the differential diagnosis with muscular dystrophies (dystrophic processes usually present muscle fat replacement that preserves muscle volume, as observed in b, c, f, g, h, and l); muscle biopsy with groups of atrophic fibres (yellow arrow in s) and hypetrophic fibres (black arrow in s), with predominance of hypertrophic type 1 fibres (red asterisk * in t) with groups of atrophic type 2 fibres (green asterisk * in t). Muscle MRI of the pelvis (u), thighs (v), and legs (w) and muscle biopsy (x and y) of a 54 years old male patient with Oculopharyngeal muscular dystrophy molecularly confirmed by two alleles with 11 GCN (GA) repetitions in the PABPN1 gene (reference value below 10 GCN repetitions for the rare autosomal recessive presentation of this usually autosomal dominant disease); muscle imaging with muscle fat replacement of the adductor magnus (green asterisk * in v), and soleus (yellow asterisk * in w), muscle biopsy with rimmed vacuoles (yellow arrow in x), and 7.5 to 10 nm (mean outer diameter of 8.5 nm) nuclear inclusions disposed in tangles and palisades (yellow arrow in y). Muscle MRI of the pelvis (z), thighs (α), and legs (β) and muscle biopsy (γ and δ) of a 61 years old female patient with mitochondriopathy of the subtype Progressive External Ophthalmoplegia (PEO) molecularly confirmed by the a deletion in the mitochondrial DNA detected in muscle tissue with 46% of heteroplasmy in Southern blot and decreased enzymatic activity of the complexes I and IV of the mitochondrial respiratory chain in muscle biopsy tissue; muscle imaging demonstrated mild abnormalities in the gluteus minimus (green asterisk * in z), and sartorius (yellow asterisk * in α), muscle biopsy with ragged red equivalent/ ragged blue fibres (yellow arrows in γ and δ) characterized by subsarcolemmal and intermyofibrillary mitochondrial proliferation combined with a dissociated "ragged" appearance of the sarcoplasm that may be suspected by the amphophilic and basophilic granular subsarcolemmal depositis around the fibre and close to the basophilic nucleus on HE (γ), and later confirmed by histochemical oxidative studies such as SDH (δ), and COX (data not shown). Muscle imaging (a, b, c, f, g, h, k, l, m, p, q, r, u, v, w, z, α, and β) axial T1-weighted magnetic resonance imaging, yellow R: right side, yellow L: left side. Muscle biopsy (d, e, i, j, n, o, s, t, x, y, γ, and δ). d HE 200x, e Transmission Electron Microscopy 3,000x, i HE 200x, j, Immunohistochemistry antibody anti-dystrophin (N-terminus) NCL-DYS3 200x (patient) and inset (control), n HE 200x, o Acid phosphatase 200x, s HE 200x, t ATPase pH 9.4 50x, x HE 400x, y Transmission Electron Microscopy 20,000x, γ HE 400x, and δ SDH 100x)

Myotonic Dystrophies (Figs. 4, 7, and 12) (Catalli et al. 2010; Udd and Krahe 2012; Wenninger et al. 2018; Gutiérrez Gutiérrez et al. 2020; Hamel 2022) are are autosomal dominant diseases caused by expansions in non-coding regions of DMPK (type 1), and CNBP (type 2) genes. This results in RNA toxicity due to gain-of-function of several proteins, responsible for the multisystemic phenotype. Steinert´s myotonic dystrophy type 1 presents a prevalence of 10.40/100,000 (Table 1) (Norwood et al. 2009). The molecular diagnosis is performed through Polymerase chain reaction (PCR) or Southern blot for myotonic dystrophy type 1; and Southern blot, long range PCR, or tetrapled primed PCR for myotonic dystrophy type 2 (Catalli et al. 2010; Udd and Krahe 2012; Wenninger et al. 2018; Gutiérrez Gutiérrez et al. 2020; Hamel 2022). Muscle biopsy is not necessary for the diagnosis, but when performed for differential diagnosis, patients with Steinert´s myotonic dystrophy type 1 may present nuclear internalization, ring fibres, and sarcoplasmic masses (Fig. 12).

Dystrophinopathies (Figs. 4, 8, and 12) (Zheng et al. 2015; Araujo et al. 2018, 2023; Viggiano et al. 2023) are X-linked muscular dystrophies related to pathogenic abnormalities in the DMD gene. From the clinical point of view, they are classified as Duchenne Muscular Dystrophy in the most severe end of the spectrum, and as Becker Muscular Dystrophy in the mild presentation. The prevalence of Duchenne muscular dystrophy (DMD gene) subtype of dystrophinopathy is 8.29/100,000 (Table 1) (Norwood et al. 2009; Araujo et al. 2018), and the prevalence of Becker muscular dystrophy (DMD gene) subtype of dystrophinopathy is 7.29/100,000 (Table 1) (Norwood et al. 2009).

Dystrophin is a structural protein that is fundamental for the integrity of the muscle sarcolemmal membrane. During the mechanical stress of muscle contraction, it forms a bridge between the sarcolemma, and actin (Viggiano et al. 2023). In about 71.2% of the patients there are great deletions in the DMD gene, duplications in 9.7%, and point mutations in 14.9% (Araujo et al. 2018, 2023; Viggiano et al. 2023). Therefore the diagnostic confirmation may be performed by molecular studies in about 95.8% of the patients: 71.2% with deletions detectable with PCR (Polymerase Chain Reaction) or Multiplex Ligation-dependent Probe Amplification (MLPA) studies, 9.7% with duplications detectable with MLPA studies, and 14.9% with point mutations detectable with Next Generation Sequencing studies. Thus muscle biopsy (Fig. 12) is strictly necessary for diagnostic confirmation of dystrophinopathy nowadays in about 4.2% of the patients.

Duchenne muscular dystrophy multiprofessional rehabilitation treatment has been well established (Araujo et al. 2018, 2023). There is great enthusiasm for the early diagnosis of Duchenne muscular dystrophy after the advent of new therapies such as exon-skipping agents nonsense read-through therapies designed for patients with mutations in exons 51 or 53 or 45 of the DMD gene (Araujo et al. 2018, 2023). Therefore, nowadays the existing approved therapies apply to only a small percentage of Duchenne Muscular Dystrophy patients but the future is promising. There are ongoing clinical trials of gene therapy with viral vectors with smaller DMD gene versions (Araujo et al 2023).

Facioscapulohumeral Muscular Dystrophy (FSHD) (Figs. 4, 9, and 12) (Lamperti et al. 2010; Ricci et al. 2016; Mul 2022; Pasnoor and Dimachkie 2019) presents a prevalence of 3.95/100,000 (Table 1) (Norwood et al. 2009; Mul 2022). In 95% of the patients, i.e., Facioscapulohumeral Muscular Dystrophy type 1 (FSHD1), it is an autosomal dominant inherited disease with a D4Z4 repeat contraction that results in derepression of the DUX4 gene that is a normal gene for the fetus, but toxic to adults. The DUX4 gene toxicity includes the induction of apoptosis, inhibition of myogenesis, and degradation of proteins, among others (Tawil 2018). Other 5% of the patients, present normal number of D4Z4 repeats and hypomethylation DUX4 gene derepression that may be combined with abnormalities in SMCHD1 (FSHD2, autosomal dominant or recessive), DNMT3B (autosomal dominant/ digenic), or LRIF1 (FSHD3, autosomal recessive/ digenic) genes (Benarroch et al. 2024; Hamanaka et al. 2020; Jia et al. 2021; Norwood et al. 2009; Mul 2022) or in cis duplications (i.e., in the same allele) of the D4Z4 repeat (Lemmers et al. 2024).

Patients with Facioscapulohumeral Muscular Dystrophy usually present the triad of: facial weakness, scapular winging, and asymmetric limb muscle weakness. Facial weakness is sometimes difficult to notice and it may manifest as transverse smile, sleeping with the eyes partially opened, inability to pucker, whistle, or drink from a straw. Absent palpebral ptosis and spared extraocular muscles are an important clues for the differential diagnosis with mitochondrial myopathies, as ophthalmoparesis is a cardinal feature of the Mitochondrial myopathy of the subtype Progressive External Ophthalmoplegia. Scapular winging is caused by weakness of the serratus anterior, and trapezius muscles with inability to lift the arms, combined with wasting of the triceps brachialis and biceps brachialis muscles, resulting in “Popeye” arms (reference to the cartoon character) with sparing of the forearm muscles (Tawil 2018; Mul 2022). Muscle biopsy is not necessary for the diagnosis and it may present nonspecific findings such as atrophy, nuclear clumps (Fig. 12), nuclear internalization in about 12.7% of the fibres, core-like areas in oxidative reactions such nicotinamide adenine dinucleotide (NADH), type 1 fibre hypertrophy, regeneration, abnormal sarcolemmal and sarcoplasmic expression of the major histocompatibility complex I (MHC-I/ HLA-ABC) in 72% of the patients, abnormal deposits reactive of the membrane attack complex (MAC/ C5b9) in 32% of the patients, and areas of decreased capillary density observed on CD31 (Hubregtse et al. 2024).

Spinal muscular atrophy or SMA (Spinal Muscular Atrophy) or 5q-Spinal Muscular Atrophy (Figs. 4, 10, and 12) (Arnold and Fischbeck 2018; Astrea et al. 2022; Oskoui and Servais 2023) is a primary neurogenic disease that affects the Lower Motor Neuron in the Anterior Horn of the Spinal Cord (Arnold and Fischbeck 2018; Oskoui and Servais 2023) (Fig. 2). It is included in this myopathy section for several reasons: (1) its high prevalence among neuromuscular disorders estimated in 1.87/100,000 (Table 1) (Norwood et al. 2009; Verhaart et al. 2017; Day et al. 2022); (2) its capacity to clinically simulate myopathies with proximal muscle weakness, and decreased deep tendon reflexes, specially in its milder forms, and difficult to diagnose in health services where neurophysiological studies such as electroneuromyography are unavailable; (3) the recent advent of disease-modifying treatments that, if administered in the first months of life, may change the course of the disease from fatal to almost normal life (Oskoui and Servais 2023). It is caused in 96% of the patients by a homozygous deletion of the SMN1 (Survival Motor Neuron) gene that causes a loss of function of the SMN protein that is necessary for RNA metabolism, autophagy, endocytosis, cell signaling, and DNA repair (Oskoui and Servais 2023). The diagnosis is confirmed by molecular exams. Muscle biopsy is not necessary for diagnosis but, when performed, it may present large groups of type 1 hypertrophic fibres (Dubowitz et al. 2020), and groups of small angulated fibres (Fig. 12).

Oculopharyngeal muscular dystrophy (Figs. 4, 11, and 12) (Yamashita et al., 2021; Stevanovski et al. 2022; Kumutpongpanich et al. 2021; Ogasawara et al. 2020, 2022; Gorman et al. 2015; Gusic and Prokisch 2021) is a late-onset myopathy characterized by progressive ptosis, dysphagia, and proximal limbs weakness (Yamashita 2021). Its prevalence is estimated in 1.0/100,000 (Table 1) (Deenen et al. 2015; Yamashita 2021). It is related to an abnormal expansion of GCN repeats. Nowadays, the diagnosis of expansion repeats is still difficult for some laboratories. It is believed that maybe the development of new technologies such as programmable targeted nanopore sequencing may enhance the diagnosis of tandem repeat expansion disorders (Stevanovski et al. 2022). Oculopharyngeal muscular dystrophy and Oculopharyngodistal myopathy share some clinical features such as ptosis, dysphagia, and dysarthria. Nevertheless, in oculopharyngodistal myopathy, muscle weakness is predominantly distal involving gastrocnemius and soleus muscles (Kumutpongpanich et al. 2021). Oculopharyngodistal myopathy may present p62 intranuclear inclusions positive in immunohistochemistry of muscle that has not been described in oculopharyngeal muscular dystrophy (Ogasawara et al. 2020, 2022).

Mitochondrial disorders (Figs. 4, 11, and 12) form a heterogeneous group of multisystemic disorders with episodes of exacerbation that affect high metabolism organs such as the brain, eyes, and muscles. The combined prevalence of all primary mitochondrial disorders was estimated in 5.0/100,000 (Table 1) (Gorman et al. 2015; Gusic and Prokisch 2021). Mitochondriopathies are genetic disorders related to pathogenic variants in more than 340 different genes (Gusic and Prokisch 2021). They are caused by defects in oxidative phosphorylation (Gusic and Prokisch 2021). The prompt diagnosis of primary defects of the biosynthesis of Coenzyme Q10 is important because oral supplementation of Coenzyme Q10 may improve considerably patients´ quality of life (Cotta et al. 2020). Next generation sequencing-based molecular methods can nowadays sequence the entire mitochondrial DNA (mtDNA), 19 nuclear encoded mitochondrial genes, and spare many patients from the invasive procedure of muscle biopsy (Gusic and Prokisch 2021; Tarnopolsky 2022). Nevertheless, several patients even after molecular studies may need a muscle biopsy for a complete morphological, histochemical, enzymatic, ultrastructural, and molecular studies in muscle tissue. This occurs because DNA extracted from muscle may avoid false-negative serum mtDNA results (Tarnopolsky 2022). Mitochondrial respiratory chain enzymatic studies may be performed in tissue from muscle biopsy to support the diagnosis of mitochondriopathy (Cotta et al. 2021).

Muscle biopsy is important in neuromuscular investigation of genetic muscular diseases, but its role has changed from obligatory first step to most frequently as a confirmatory phenotypic exam.

Conclusion

With the advent of new technologies, myopathology practice has expanded to much more than muscle biopsy interpretation. A multiprofessional integrated approach is essential for precise neuromuscular diagnosis. Detailed clinical examination with the formulation of phenotypic hypothesis is the basis for appropriate diagnosis in the Surgical-Molecular Pathology era. Clinical findings guide the use of appropriate ancillary studies.

Promising genetic therapies have emerged in the last years. Therefore, clinical, epidemiological, neurophysiological, laboratorial, imaging, molecular, and physiopathologic aspects are essential for adequate neuromuscular diagnosis and treatment.