Essential neuromuscular advice for pathologists: Limb Girdle Muscular Dystrophy (second of two parts)

Abstract

Background

Limb Girdle Muscular Dystrophy is defined as a group of progressive autosomal recessive (85%, 28 genes) and autosomal dominant (15%, 5 genes) muscular dystrophies described in at least two unrelated families, affecting individuals that achieve independent walking, with predominant proximal muscles weakness at presentation, elevated serum creatine kinase activity, dystrophic changes on muscle biopsy, and degeneration on muscle imaging over the course of the disease.

Main body

The aims of this review are: (1) to show the recent Limb Girdle Muscular Dystrophy (LGMD) genetic classification illustrated with clinical and physiopathological characteristics, and cellular localization of the main gene products; (2) to present muscle radiophenotypes with an algorithm for differential diagnosis; (3) to show the role of muscle biopsy for phenotypic characterization, and pathogenicity confirmation in the era of surgical-molecular pathology.

Conclusion

Pathologists may be aware of clinical, neurophysiological, laboratorial, imaging, molecular, and muscle biopsy modalities to provide a precise phenotypic-genotypic diagnosis for adequate rehabilitation care, and genetic counselling.

Background

Muscular Dystrophy is a muscle disease clinically characterized by progressive muscle weakness. Morphologically, it presents dystrophic features on muscle biopsy, characterized by distorted architecture, variation in muscle fibre calibre, necrosis, phagocytosis, and regeneration (Cotta et al. 2014a, b).

Limb Girdle Muscular Dystrophy (LGMD) 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, elevated serum creatine kinase activity, dystrophic changes on muscle biopsy, and degeneration on muscle imaging over the course of the disease. The dystrophic (histological) pattern is characterized by abnormal architecture with endomysial fibrosis that results in fibrous replacement of the muscle tissue followed by muscle fat replacement with striking variation in fibre calibre with hypertrophy, atrophy; hypertrophic fibres may present fibre splitting and abnormally internalized nuclei; the driver physiopathological events that result in the dystrophic pattern are necrosis, phagocytosis, and regeneration (Cotta et al. 2024b).

The diagnostic evaluation of LGMD patients starts with the phenotypic characterization, first with the exclusion of treatable, and more common neuromuscular disorders. The LGMD diagnosis is confirmed by molecular studies such as Next Generation Sequencing. Muscle biopsy is performed in cases of inconclusive genetic studies, suspicion of autoimmune myositis, or other differential diagnosis.

Brief overview of the Limb Girdle Muscular Dystrophy genetic classification

Limb Girdle Muscular Dystrophy (LGMD) has been classified in five dominant and 28 recessive subtypes (Straub et al. 2018; Benarroch et al. 2024). The dominant forms are classified as LGMD D and the recessive forms as LGMD R. They are sub-classified with numbers following the chronological order of the discovery of the genetic sites that contain the mutated gene.

The current and the old genetic classification of LGMD with a description of the gene, protein locus and protein function is presented in Fig. 1 (Benarroch et al. 2024; Cotta et al. 2014a, b; Straub et al. 2018), and Table 1 (Argente-Escrig et al. 2021; Arvio et al. 2019; Beckmann and Spencer 2008; Benarroch et al. 2024; Bonnemann 2011; Cenacchi et al. 2013; Chompoopong and Milone 2023; Coppens et al. 2021; Dai et al. 2019; De Ridder et al. 2023; Endo et al. 2015; Finsterer 2018; Gaertner et al. 2022; Geis et al. 2019; Guan et al. 2023; Huang et al. 2023; Lampe and Bushby 2005; Li et al. 2023; Lv et al. 2021; Mahmood et al. 2023; Melia et al., 2013; Meyer et al. 2022; Morales-Rosado et al. 2023; Mroczek et al. 2020; Munot et al. 2022; Murphy and Straub 2015; Palenzuela et al. 2003; Panicucci et al. 2023; Saenz et al., 2005; Sandell et al. 2016; Savarese et al. 2020; Soontrapa and Liewluck 2022; Starling et al. 2004; Straub et al. 2018; Vainzof et al. 2021; van Tol et al. 2019; Vargas‐Franco et al., 2022; Vieira et al. 2014; Wang et al. 2022; Xie et al. 2019; Yang et al. 2021; Yogev et al. 2023; Zatz and Starling 2005; Zhou et al. 2022).

Fig. 1

Schematic diagram of muscle gene products involved in each subtype of Limb Girdle Muscular Dystrophy (LGMD) according to the most recent genetic classification (Benarroch et al. 2024; Cotta et al. 2014a, b; Straub et al. 2018). Letters R1 to R28 and D1 to D5 indicate, respectively, autosomal recessive LGMD R1 to LGMD R28, and autosomal dominant LGMD D1 to LGMD D5. A muscle fibre is represented with the contractile apparatus of the sarcomere, muscle membrane, extracellular matrix, nucleus, sarcoplasm containing the Golgi complex, and endoplasmic reticulum. Nine different LGMD have been classified in a subgroup as Muscular dystrophy dystroglycanopathy (MDDG): R9, R11, R13, R14, R15, R16, R19, R20, and R24. In green letters are represented the most common LGMD (Winckler et al. 2019): calpain (R1), dysferlin (R2), sarcoglycan complex proteins alpha (R3), beta (R4), gamma (R5), and delta (R6), fukutin related protein (FKRP) (R9), telethonin (R7), and anoctamin (R12)

Table 1 Limb Girdle Muscular Dystrophy (LGMD) classification (Argente-Escrig et al. 2021; Arvio et al. 2019; Beckmann and Spencer 2008; Benarroch et al. 2024; Bonnemann 2011; Cenacchi et al. 2013; Chompoopong and Milone 2023; Coppens et al. 2021; Dai et al. 2019; De Ridder et al. 2023; Endo et al. 2015; Finsterer 2018; Gaertner et al. 2022; Geis et al. 2019; Guan et al. 2023; Huang et al. 2023; Lampe and Bushby 2005; Li et al. 2023; Lv et al. 2021; Mahmood et al. 2023; Melia et al., 2013; Meyer et al. 2022; Morales-Rosado et al. 2023; Mroczek et al. 2020; Munot et al. 2022; Murphy and Straub 2015; Palenzuela et al. 2003; Panicucci et al. 2023; Saenz et al., 2005; Sandell et al. 2016; Savarese et al. 2020; Soontrapa and Liewluck 2022; Starling et al. 2004; Straub et al. 2018; Vainzof et al. 2021; van Tol et al. 2019; Vargas‐Franco et al., 2022; Vieira et al. 2014;Wang et al. 2022; Xie et al. 2019; Yang et al. 2021; Yogev et al. 2023; Zatz and Starling 2005; Zhou et al. 2022)

A brief description of the most peculiar clinical characteristics of each subtype of LGMD, the age of onset, and serum creatine kinase levels are provided in Table 2 (Alonso-Pérez et al. 2020; Alonso-Pérez et al. 2022; Argente-Escrig et al. 2021; Arvio et al. 2019; Astrea et al. 2018; Baranello et al. 2015; Belaya et al. 2015; Bello et al. 2012; Berardo et al 2019; Bobadilla-Quesada et al. 2020; Bögershausen et al. 2013; Boito et al. 2005; Bönnemann 2011; Brun et al. 2018; Cenacchi et al. 2013; Cerino et al. 2020; Chandrasekharan et al. 2021; Chen et al. 2021; Cheng et al. 2022; Chompoopong & Milone 2023; Christiansen et al 2022; Cirak et al. 2013; Clement et al. 2008; Coppens et al. 2021; Cossée et al. 2009; Costa et al. 2020; Costa et al. 2022; Cotta et al. 2014b; Dai et al. 2019; De Ridder et al. 2023; Dinçer et al. 2003; Dong et al. 2015; Endo et al. 2015; Estephan et al. 2022; Fernández-Eulate et al. 2020; Finsterer 2018; Fiorillo et al. 2013; Gaertner et al. 2022; Ganaraja et al. 2021; Geis et al. 2019; González-Mera et al. 2021; Guan et al. 2023; Gundesli et al. 2010; Haberlova et al. 2014; Hadouiri et al. 2021; Halmo et al. 2017; Hara et al. 2011; Harris et al. 2017a, Harris et al. 2017b; Hicks et al. 2011; Huang et al. 2023; Jensen et al. 2015; Jensen et al. 2023; Johnson et al. 2019; Justel et al. 2023; Koss-Harnes et al. 2004; Kubota et al. 2018; Kwong et al. 2023; Lampe and Bushby 2005; Larson et al. 2018; Libell et al. 2020; Løkken et al. 2015; Lv et al. 2023; Magri et al. 2020; Mahmood et al. 2023; Malfatti & Richard 2020; Marchuk et al. 2021; Melià et al. 2013; Meyer et al. 2022; Morales-Rosado et al. 2023; Moreira et al. 1997; Moreira et al. 2000; Mroczek et al. 2020; Munot et al. 2022; Murphy et al. 2020; Negrão et al. 2010; Nguyen et al. 2007; Nicolau et al. 2019; Oestergaard et al. 2016; Paim et al. 2013; Palenzuela et al. 2003; Panicucci et al. 2023; Poppe et al. 2003; Riisager et al. 2013; Rosales et al. 2010; Saccone et al. 2008; Sandell et al. 2016; Savarese et al. 2020; Schindler et al. 2016; Servián-Morilla et al. 2016; Servián-Morilla et al. 2020; Silva et al. 2019; Soontrapa & Liewluck 2022; Starling et al. 2004; Sun et al. 2020; Swan et al. 2023; Tasca et al. 2013; Tian et al. 2019; Torella et al. 2013; Udd 1992; Vainzof et al. 2021; van Tol et al. 2019; Vieira et al. 2014; Vihola et al. 2018; Villar Quiles et al. 2020; Vissing et al. 2019; Wang et al. 2022; Willis et al., 2022; Yogev et al. 2023; Zanoteli et al. 2020; Zhang et al. 2022).

Table 2 Peculiar characteristics of recessive (LGMD R) and dominant (LGMD D) Limb Girdle Muscular Dystrophy subtypes (Alonso-Pérez et al. 2020; Alonso-Pérez et al. 2022; Argente-Escrig et al. 2021; Arvio et al. 2019; Astrea et al. 2018; Baranello et al. 2015; Belaya et al. 2015; Bello et al. 2012; Benarroch et al. 2024; Berardo et al 2019; Bobadilla-Quesada et al. 2020; Bögershausen et al. 2013; Boito et al. 2005; Bönnemann 2011; Brun et al. 2018; Cenacchi et al. 2013; Cerino et al. 2020; Chandrasekharan et al. 2021; Chen et al. 2021; Cheng et al. 2022; Chompoopong & Milone 2023; Christiansen et al 2022; Cirak et al. 2013; Clement et al. 2008; Coppens et al. 2021; Cossée et al. 2009; Costa et al. 2020; Costa et al. 2022; Cotta et al. 2014b; Cotta et al. 2024a; Dai et al. 2019; De Ridder et al. 2023; Dinçer et al. 2003; Dong et al. 2015; Endo et al. 2015; Estephan et al. 2022; Fernández-Eulate et al. 2020; Finsterer 2018; Fiorillo et al. 2013; Gaertner et al. 2022; Ganaraja et al. 2021; Geis et al. 2019; González-Mera et al. 2021; Guan et al. 2023; Gundesli et al. 2010; Haberlova et al. 2014; Hadouiri et al. 2021; Halmo et al. 2017; Hara et al. 2011; Harris et al. 2017a, Harris et al. 2017b; Hicks et al. 2011; Huang et al. 2023; Jensen et al. 2015; Jensen et al. 2023; Johnson et al. 2019; Justel et al. 2023; Koss-Harnes et al. 2004; Kubota et al. 2018; Kwong et al. 2023; Lampe and Bushby 2005; Larson et al. 2018; Libell et al. 2020; Løkken et al. 2015; Lv et al. 2023; Magri et al. 2020; Mahmood et al. 2023; Malfatti & Richard 2020; Marchuk et al. 2021; Melià et al. 2013; Meyer et al. 2022; Morales-Rosado et al. 2023; Moreira et al. 1997; Moreira et al. 2000; Mroczek et al. 2020; Munot et al. 2022; Murphy et al. 2020; Negrão et al. 2010; Nguyen et al. 2007; Nicolau et al. 2019; Oestergaard et al. 2016; Paim et al. 2013; Palenzuela et al. 2003; Panicucci et al. 2023; Poppe et al. 2003; Riisager et al. 2013; Rosales et al. 2010; Saccone et al. 2008; Sandell et al. 2016; Savarese et al. 2020; Schindler et al. 2016; Servián-Morilla et al. 2016; Servián-Morilla et al. 2020; Silva et al. 2019; Soontrapa & Liewluck 2022; Starling et al. 2004; Straub et al. 2018; Sun et al. 2020; Swan et al. 2023; Tasca et al. 2013; Tian et al. 2019; Torella et al. 2013; Udd 1992; Vainzof et al. 2021; van Tol et al. 2019; Vieira et al. 2014; Vihola et al. 2018; Villar Quiles et al. 2020; Vissing et al. 2019; Wang et al. 2022; Willis et al., 2022; Yogev et al. 2023; Zanoteli et al. 2020; Zhang et al. 2022)

Localization of the main Limb Girdle Muscular Dystrophy gene products

The LGMD gene products related to each subtype may be related to the sarcolemmal membrane, nucleus, sarcomere, Golgi complex, endoplasmic reticulum, and extracellular matrix (Fig. 1) (Benarroch et al. 2024; Cotta et al. 2014a; Straub et al. 2018). Knowledge of the exact localization of these proteins is very important for diagnosis. Pathologists may use antibodies to detect total or partial deficiency in the expression of these proteins to confirm the phenotype, e.g. anti-dysferlin antibody reaction is observed in the sarcolemmal membrane whereas anti-telethonin antibodies react with the sarcommeric protein telethonin (intrasarcoplasmic/ intracytoplasmatic reaction). Please see below the sections “Clinical-radiological-pathological approach for the diagnosis of Limb Girdle Muscular Dystrophy”, and "Use of muscle biopsy for Limb Girdle Muscular Dystrophy phenotypic characterization".

Frequency of Limb Girdle Muscular Dystrophy subtypes and diagnostic yield

The most frequent LGMD subtypes are calpainopathies (LGMD R1 calpain-related, former LGMD2A), dysferlinopathies (LGMD R2 dysferlin-related, former LGMD2B), sarcoglycanopathies (LGMD R3 alpha sarcoglycan-related, LGMD R4 beta-sarcoglycan-related, LGMD R4 gamma sarcoglycan-related, and LGMD R5 delta sarcoglycan-related, respectively former LGMD2D, LGMD2E, LGMD2C, and LGMD2F), FKRP-pathies (LGMD R9 FKRP-related, former LGMD2I), telethoninopathies (LGMD R7 telethonin-related, former LGMD2G), anoctaminopathies (LGMD R12 anoctamin 5 -related, former LGMD2L), and collagenopathies 6 (LGMD R22 collagen 6-related/ LGMD D5 collagen 6-related, Bethlem myopathy). The relative frequency of the most common LGMD subtypes is presented in Table 3 (Bevilacqua et al. 2020; Chakravorty et al. 2020; Cotta et al. 2017; Fanin et al. 2009; Gomez-Diaz et al., 2012; Guglieri et al. 2008; Lo et al. 2008; Lorenzoni et al. 2023; Liu et al. 2019; Norwood et al. 2009; Winckler et al. 2019).

Table 3 Estimated prevalence and relative frequencies of the most common LGMD subtypes (Bevilacqua et al. 2020; Chakravorty et al. 2020; Cotta et al. 2017; Fanin et al. 2009; Gomez-Diaz et al., 2012; Guglieri et al. 2008; Lo et al. 2008; Lorenzoni et al. 2023; Liu et al. 2019; Norwood et al. 2009; Winckler et al. 2019)

The precise diagnosis of the specific subtype of Limb Girdle Muscular Dystrophy is not straightforward and the diagnostic yield of different methods, i.e., the detection rate, varies in several reference centres from around 33.8% to 68.3% as presented in Table 4 (Bevilacqua et al. 2020; Chakravorty et al. 2020; Cotta et al. 2017; Lorenzoni et al. 2023; Özyilmaz et al., 2019; Reddy et al., 2017; Winckler et al. 2022; Yu et al. 2017). Therefore, even in reference tertiary centres around the world, about one third of all LGMD patients may remain with a phenotypic clinical diagnosis of Limb Girdle Muscular Dystrophy, confirmed by the dystrophic pattern on muscle biopsy, without knowledge of the culprit gene, or defective protein. A diagnostic yield above 60% was reported with the combined use of molecular and myopathological results (Lorenzoni et al. 2023; Yu et al. 2017).

Table 4 Diagnostic yield of Limb Girdle Muscular Dystrophy (LGMD) diagnosis with different methods from 2017 to 2023 (Bevilacqua et al. 2020; Chakravorty et al. 2020; Cotta et al. 2017; Lorenzoni et al. 2023; Özyilmaz et al., 2019; Reddy et al., 2017; Winckler et al. 2022; Yu et al. 2017)

Limb Girdle Muscular Dystrophy muscle radiophenotypes for differential diagnosis

Muscle imaging has evolved as a very useful tool for phenotypic LGMD characterization (Figs. 2, 3, 4, 5, 6, and 7) (Aivazoglou L et al. 2022; Argente‐Escrig et al., 2021; Baranello et al. 2015; Belaya et al. 2015; Berardo et al. 2019; Bohlega et al. 2018; Cerino et al. 2020; Chandrasekharan et al. 2021; Chompoopong and Milone 2023; Cirak et al. 2013; Clement et al. 2008; Coppens et al. 2021; Costa et al. 2022; Cotta et al. 2023; Cotta et al., unpublished data; Díaz‐Manera et al., 2021; Domínguez-González et al. 2022; Dong et al. 2015; Endo et al. 2015; Fu et al. 2016; González-Mera et al. 2021; Haberlova et al. 2014; Hafner et al. 2014; Harris et al. 2017a; Harris et al. 2017b; Hicks et al. 2011; Jonson et al. 2018; Johnson et al. 2019; Kim et al. 2018; Kojima et al. 2017; Kristiansen et al. 2023; Larson et al. 2018; Liang et al. 2015; Llansó et al. 2023; Malfatti et al. 2020; Melià et al. 2013; Meyer et al. 2022; Morales-Rosado et al. 2023; Mroczek et al. 2020; Munot et al. 2022; Nam et al. 2015; Palmio et al. 2015; Panicucci et al. 2023; Pitceathly et al. 2013; Riisager et al.. 2013; Rodriguez et al. 2023; Sandell et al. 2010; Sandell et al. 2013; Savarese et al. 2020; Servián-Morilla et al. 2016; Servián-Morilla et al. 2020; Silva et al. 2019; Spinazzi et al. 2021; Straub et al. 2012; Suárez et al. 2018; Sun et al. 2019; Swan et al. 2023; Tasca et al. 2013; Tasca et al. 2017; Tasca et al. 2018; Tsai et al. 2017; Vicente et al. 2020; Villar Quiles et al. 2020; Vissing et al. 2016; Vissing et al. 2019; Yang et al. 2021; Yogev et al. 2023; Wattjes et al. 2010; Zima et al. 2020).

Fig. 2

Muscle imaging grading (Wattjes et al. 2010) diagrams of LGMD R1 (a, b, and c), LGMD R2 (d, e, and f), LGMD R3 (g, h, and i), LGMD R4 (j, k, and l), LGMD R5 (m, n, and o), LGMD R6 (p, q, and r), LGMD R7 (s, t, and u), and LGMD R8 (v, w, and x) of the pelvis (a, d, g, j, m, p, s, and v), thighs (b, e, h, k, n, q, t, and w), and legs (c, f, i, l, o, r, u, and x). LGMD R1 CAPN3 (Straub et al 2012; Aivazoglou L et al. 2022) (a, b, and c). a Muscle fat replacement is severe in gluteus minimus, moderate of gluteus medius, mild of gluteus maximus. b Severe in adductor magnus, biceps femoris long head, semitendinosus, semimembranosus, mild in gracilis, and vastus intermedius. c Severe in soleus, and gastrocnemius medialis. LGMD R2 DYSF (Straub et al 2012; Llansó et al 2023) (d, e, and f) d Pelvis with severe involvement of the gluteus minimus and mild involvement of the gluteus maximus. e Thighs with moderate involvement of the vastus lateralis, vastus medialis, adductor magnus, adductor longus, biceps femoris long head, semitendinosus, semimembranosus, and mild involvement of rectus femoris, vastus intermedius, sartorius and gracilis. f Legs with global decreased volume (calf atrophy), moderate involvement of soleus, gastrocnemius medialis, and gastrocnemius lateralis with mild involvement of tibialis anterior, extensor group, and peroneus group. Sarcoglycanopathy group (Straub et al 2012; Tasca et al 2018): LGMD R3 (g, h, and i), LGMD R4 (j, k, and l), LGMD R5 (m, n, and o), LGMD R6 (p, q, and r) LGMD R3 SGCA g Pelvis with severe muscle fat replacement of the gluteus minimus, and gluteus medius. h Thighs with compensatory hypertrophy of the rectus femoris, sartorius, and gracilis, severe muscle fat replacement of the vastus intermedius, biceps femoris long head, and adductor magnus, moderate involvement of vastus medialis, adductor longus, and semimembranosus. i. Legs with preservation of all muscle groups and true calf muscle hypertrophy (diameter of the calves almost the same as the thighs). LGMD R4 SGCB j Pelvis with severe muscle fat replacement of the gluteus minimus. k Thighs with compensatory hypertrophy of the rectus femoris, sartorius, and gracilis, severe muscle fat replacement of the vastus intermedius, and adductor magnus, moderate involvement of vastus medialis, adductor longus, biceps femoris long head, and semimembranosus. l. Legs with preservation of all muscle groups and true calf muscle hypertrophy (diameter of the calves almost the same as the thighs). LGMD R5 SGCG m Pelvis with severe muscle fat replacement of the gluteus minimus, gluteus medius, and gluteus maximus. n Thighs with compensatory hypertrophy of the sartorius, and gracilis, severe muscle fat replacement of the adductor magnus, moderate involvement of vastus intermedius, vastus medialis, adductor longus, and biceps femoris long head. o. Legs with slight involvement of all muscle groups and true calf muscle hypertrophy (diameter of the calves almost the same as the thighs). LGMD R6 SGCD p Pelvis with severe muscle fat replacement of the gluteus minimus, gluteus medius and gluteus maximus. q Thighs with severe muscle fat replacement of the adductor magnus, quadriceps femoris (i.e. vastus lateralis, vastus medialis, vastus intermedius, and rectus femoris), biceps femoris long head, semitendinosus, and semimembranosus. r. Legs with severe involvement of gastrocnemius medialis, gastrocnemius lateralis, peroneus group, moderate muscle fat replacement of the tibialis anterior, and extensor groups, and mild involvement of the soleus with pseudohypertrophy of the calf (muscle fat replacement with diameter of the calves almost the same as the thighs). LGMD R7 TCAP (Cotta et al. 2024a) (s, t, and u) s Pelvis with severe muscle fat replacement of gluteus maximus, moderate involvement of gluteus medius, and gluteus minimus. t Thighs with asymmetric involvement, in this particular example with semitendinosus severely affected in the right side and moderately replaced in the left side; severe involvement of adductor magnus, biceps femoris long head, and semimembranosus; moderate involvement of quadriceps femoris (vastus lateralis, vastus medialis, vastus intermedius, and rectus femoris) with compensatory hypertrophy of sartorius, and gracilis. u Legs with asymmetric muscle fat replacement that in this particular example it is severe in right tibialis anterior, and moderate in left tibialis anterior; moderate muscle fat replacement of gastrocnemius medialis, and mild involvement of soleus. LGMD R8 TRIM32 (Johnson et al. 2019; Chandrasekharan et al. 2021) (v, w, and x). v Pelvis with severe muscle fat replacement of gluteus maximus, gluteus medius, and gluteus minimus. w Thighs with severe muscle fat replacement of the medial compartment involving sartorius, adductor longus, and adductor magnus with relative preservation of the gracilis, and severe involvement of the posterior compartment, mostly biceps femoris long head, and semimembranosus, with relative preservation of the semitendinosus. x Legs with severe muscle fat replacement of the posterior compartment involving the soleus, gastrocnemius medialis, and gastrocnemius lateralis

Fig. 3

Muscle imaging grading (Wattjes et al. 2010) diagrams of LGMD R9 (a, b, and c), LGMD R10 (d, e, and f), LGMD R11 (g, h, and i), LGMD R12 (j, k, and l), LGMD R13 (m, n, and o), LGMD R14 (p, q, and r), LGMD R15 (s, t, and u), and LGMD R16 (v, w, and x) of the pelvis (a, d, g, j, m, p, s, and v), thighs (b, e, h, k, n, q, t, and w), and legs (c, f, i, l, o, r, u, and x). LGMD R9 FKRP (Straub et al. 2012; Cotta et al. 2014a; Villar Quiles et al. 2020) (a, b, and c) a Pelvis with moderate muscle fat replacement of gluteus medius, and gluteus minimus. b Thighs with severe muscle fat replacement of adductor magnus, and biceps femoris long head with moderate involvement of semimembranosus, and semitendinosus with preservation of the rectus femoris. c Legs with severe muscle fat replacement of gastrocnemius medialis, gastrocnemius lateralis, soleus, and peroneus muscles. LGMD R10 TTN (Harris et al. 2017b; Savarese et al. 2020) (d, e, and f) d Pelvis with moderate muscle fat replacement of gluteus maximus, gluteus medius, and gluteus minimus. e Thighs with severe muscle fat replacement of vastus lateralis, vastus intermedius, and semitendinosus with moderate involvement of rectus femoris. f Legs with severe muscle fat replacement of soleus, peroneous group; moderate involvement of gastrocnemius medialis; tibialis anterior may be either moderatly affected (Harris et al. 2017a, Harris et al. 2017b) or with only focal changes (Savarese et al. 2020). LGMD R11 POMT1 (Haberlova et al. 2014; Hafner et al. 2014) (g, h, and i) g Pelvis with severe muscle fat replacement of gluteus maximus. h Thigh with severe muscle fat replacement of adductor magnus, and diffuse moth eaten pattern (hachurated graticule) of moderate muscle fat replacement involving vastus intermedius, vastus medialis, biceps femoris long head, semitendinosus, and semimembranosus, with compensatory hypertrophy of gracilis. i Legs with moderate muscle fat replacement of gastrocnemius medialis, extensor group, and peroneous group. LGMD R12 ANO5 (Hicks et al. 2011; Straub et al. 2012; Silva et al. 2019) (j, k, and l) j Pelvis with moderate involvement of gluteus minimus and mild involvement of gluteus maximus, and gluteus medius. k Thighs with asymmetric muscle fat replacement that, in this example, it is severe in adductor magnus, and semimembranosus, in the right side, and semitendinosus in the left side, with variable involvement of vastus lateralis, vastus intermedius, vastus medialis, and biceps femoris long head, as well as variable preservation of recuts femoris; compensatory hypertrophy of sartorius, and gracilis. l Legs with severe muscle fat replacement of gastrocnemius medialis, and moderate involvement of soleus. LGMD R13 FKTN (Riisager et al. 2013) (m, n, and o) m Pelvis with severe muscle fat replacement of gluteus maximus. n Thighs with severe involvement of adductor magnus, biceps femoris long head; moderate involvement of vastus lateralis, vastus medialis, and vastus intermedius with compensatory hypertrophy of sartorius, and gracilis. o Legs with mild involvement of soleus and gastrocnemius medialis. LGMD R14 POMT2 (Panicucci et al. 2023) (p, q, and r) p Pelvis with severe muscle fat replacement of gluteus medius, and gluteus minimus. q Thighs with severe involvement of adductor magnus, adductor longus, biceps femoris long head; moderate involvement of rectus femoris, semitendinosus, and semimembranosus; and preservation of gracilis. r Legs with moderate involvement of gastrocnemius medialis, mild involvement of gastrocnemius lateralis, and soleus. LGMD R15 POMGNT1 (Clement et al. 2008) (s, t, and u) Asterisks * indicate diagrams that were prepared based on descriptions of the exams without photographs for consultation in Clement et al. 2008. t wasting of the hamstrings (i.e. biceps femoris, semitendinosus, and semimebranosus). LGMD R16 DAG1 (Dong et al. 2015) (v, w, and x) *Diagrams were prepared based on descriptions of the exams without photographs for consultation in Dong et al. 2015. w Thighs with muscle fat replacement of rectus femoris, and semimembranosus. x Legs with involvement of gastrocnemius medialis and gastrocnemius lateralis

Fig. 4

Muscle imaging grading (Wattjes et al. 2010) diagrams of LGMD R17 (a, b, and c), LGMD R18 (d, e, and f), LGMD R19 (g, h, and i), LGMD R20 (j, k, and l), LGMD R21 (m, n, and o), LGMD R22 (p, q, and r), LGMD R23 (s, t, and u), and LGMD R24 (v, w, and x) of the pelvis (a, d, g, j, m, p, s, and v), thighs (b, e, h, k, n, q, t, and w), and legs (c, f, i, l, o, r, u, and x). LGMD R17 PLEC (Mroczek et al. 2020 and Argente‐Escrig et al. 2021) (a, b, and c) *Diagrams were prepared based on photographs and descriptions of the thighs and legs by Mroczek et al. 2020 and Argente‐Escrig et al. 2021b Thighs with muscle fat replacement of adductor magnus, adductor longus, biceps femoris, semitendinosus, and semimembranosus, with preservation of rectus femoris and sartorius, and mild compensatory hypertrophy of gracilis. c Legs with severe muscle fat replacement of tibialis anterior, and extensor group. LGMD R18 TRAPPC11 (Liang et al. 2015, Larson et al. 2018, and Munot et al. 2022) (d, e, and f) *Diagrams were prepared based on descriptions of the exams by Liang et al. 2015, Larson et al. 2018, and Munot et al. 2022. d Pelvis with muscle fat replacement of gluteus maximus, and other glutei. e Thighs with involvement of adductor magnus, and adductor longus. f Legs with involvement of the posterior compartment. LGMD R19 GMPPB (Belaya et al. 2015; Chompoopong and Milone 2023) (g, h, and i) *Diagrams were prepared based on photographs and descriptions of the thighs by Belaya et al. 2015, and only descriptions by Chompoopong and Milone 2023) g Pelvis with muscle fat replacement of gluteus maximus. h Thighs with severe involvement of adductor magnus, moderate muscle fat replacement of biceps femoris long head, biceps femoris short head, semitendinosus, and semimembranosus; mild involvement of vastus lateralis and vastus medialis with preservation of rectus femoris, sartorius, gracilis, and vastus intermedius. i Legs with severe muscle fat replacement of tibialis anterior, extensor group, and deep gastrocnemius medialis, and mild involvement of peroneus group with preservation of soleus, gastrocnemius lateralis, and tibialis posterior. LGMD R20 CRPPA (former ISPD) (Cirak et al. 2013; Tasca et al. 2013; Baranello et al. 2015; and Yang et al. 2021) (j, k, and l) *Diagrams were prepared based on photographs and descriptions of the thighs and legs by Cirak et al. 2013; Tasca et al. 2013; Baranello et al. 2015, and Yang et al. 2021. k Thighs with severe muscle fat replacement of adductor magnus, moderate involvement of anterior and posterior compartments with sparing and compensatory hypertrophy of sartorius, gracilis, biceps femoris short head, and adductor longus. l Legs with muscle fat replacement of gastrocnemius medialis deep part, gastrocnemius lateralis deep part, and peroneus group. LGMD R21 POGLUT1 (Servián-Morilla et al. 2016, 2020) (m, n, and o) *Diagrams were prepared based on photographs and descriptions of the thighs and legs by Servián-Morilla et al. 2016; Servián-Morilla et al. 2020. n Thighs with "inside to outside" pattern of muscle fat replacement that is severe in the internal parts of the muscles compared to the external areas of vastus lateralis, vastus intermedius, biceps femoris long head, semitendinosus, and semimembranosus, with severe involvement of adductor magnus, and vastus medialis with preservation of sartorius, gracilis, and adductor longus. o Legs with involvement of the soleus, deep areas of tibialis anterior, gastrocnemius medialis, and gastrocnemius lateralis. LGMD R22 COL6A1, COL6A2, COL6A3 (Straub et al. 2012; Fu et al. 2016; Suárez et al. 2018) (p, q, and r). p Pelvis with involvement of glutei muscles. q Thighs with involvement of the periphery of vastus lateralis more than the central part called "sandwich sign" or "striped pattern", a central shadow ("target sign") of involvement in rectus femoris, peripheral involvement of adductor magnus, and relative preservation of sartorius and gracilis. r Legs with peripheral involvement between the soleus, and gastrocnemius medialis, called "rim" of fatty infiltration. LGMD R23 LAMA2 (Harris et al. 2017a; Meyer et al. 2022) (s, t, and u). s Pelvis with involvement of glutei muscles that is more pronounced in gluteus medius and gluteus minimus than in gluteus maximus. t Thighs with involvement of the periphery of vastus lateralis more than the central part called "sandwich sign" or "striped pattern"; a peripheral involvement is observed in rectus femoris; moderate involvement of adductor magnus, adductor longus, and biceps femoris long head with preservation and hypertrophy of sartorius, and gracilis. u Legs may present muscle fat replacement of peroneus group, peripheral involvement between the soleus, and gastrocnemius medialis, called "rim" of fatty infiltration. LGMD R24 POMGNT2 (Endo et al. 2015) (v, w, and x). *Diagrams were prepared based on photographs of the thighs and legs by Endo et al. 2015. w Thighs with atrophy and mild muscle fat replacement of semitendinosus. x Legs with mild muscle fat replacement of peroneus group, and gastrocnemius lateralis (observed in the photographs by Endo et al. 2015)

Fig. 5

Muscle imaging grading (Wattjes et al. 2010) diagrams of LGMD R25 (a, b, and c), LGMD R26 (d, e, and f), LGMD R27 (g, h, and i), LGMD D1 (j, k, and l), LGMD D2 (m, n, and o), LGMD D3 (p, q, and r), LGMD D4 (s, t, and u), and LGMD D5 (v, w, and x) of the pelvis (a, d, g, j, m, p, s, and v), thighs (b, e, h, k, n, q, t, and w), and legs (c, f, i, l, o, r, u, and x). LGMD R25 BVES (Swan et al. 2023) (a, b, and c) a Pelvis with preservation of glutei muscles. b Thighs with severe muscle fat replacement of adductor longus, and moderate involvement of adductor magnus; there is abnormal edema (STIR sequences in photographs described by Swan et al. 2023) (data not represented) with asymmetric mild involvement of vastus lateralis (right/left), vastus intermedius (right/ left) vastus medialis (right/left), rectus femoris (left), biceps femoris (right), and semimembranosus (left) with preservation of sartorius and gracilis. c Legs with moderate symmetric muscle fat replacement of gastrocnemius medialis. LGMD R26 POPDC3 (Vissing et al. 2019) (d, e, and f) d Pelvis with moderate involvement of gluteus medius. e Thighs with end stage muscle fat replacement of adductor magnus, severe involvement of vastus lateralis, vastus intermedius, vastus medialis, biceps femoris long head, and semimembranosus; mild involvement of rectus femoris, semitendinosus, and adductor longus. f Legs with severe muscle fat replacement of gastrocnemius medialis, moderate involvement of gastrocnemius lateralis, and mild involvement of soleus. LGMD R27 JAG2 (Coppens et al. 2021) (g, h, and i) g Pelvis with moderate muscle fat replacement of gluteus maximus, gluteus medius, and gluteus minimus. h Thigh with severe muscle fat replacement of adductor magnus, adductor longus, biceps femoris, semitendinosus, and semimembranosus, with peripheral areas of preservation in semitendinosus, semimembranosus, and biceps femoris long head; in the anterior compartment there is moderate involvement of deep vastus lateralis (described by Coppens et al. 2021 as chevron sign of muscle fat replacement), vastus medialis, vastus intermedius, and deep rectus femoris; there is preservation of sartorius, and gracilis. i Legs with areas of moderate involvement of deep gastrocnemius medialis and superficial tibialis anterior, with mild involvement of soleus. LGMD D1 DNAJB6 (Sandell et al. 2010, 2013; Nam et al. 2015; Palmio et al. 2015; Kojima et al. 2017; Tsai et al. 2017; Bohlega et al. 2018; Kim et al. 2018; Jonson et al. 2018; Zima et al. 2020) (j, k, and l) *Diagrams were prepared based on descriptions of the pelvis by Sandell et al. 2013, photographs of the thighs and legs by Sandell et al. 2010, Sandell et al. 2013; Nam et al. 2015; Palmio et al. 2015; Kojima et al. 2017; Tsai et al. 2017; Bohlega et al. 2018; Kim et al. 2018; Jonson et al. 2018; and Zima et al. 2020. j Pelvis with mild muscle fat replacement of glutei muscles. k Thighs with severe involvement of the adductor magnus, biceps femoris, and semimembranosus, moderate involvement of semitendinosus and vastus intermedius; and mild involvement of vastus lateralis; rectus femoris, vastus medialis, sartorius, and gracilis are usually spared. l Legs with involvement of the posterior compartment with severe muscle fat replacement of soleus, moderate involvement of gastrocnemius medialis, and variable involvement (from mild to moderate) of gastrocnemius lateralis. LGMD D2 TNPO3 (Melià et al. 2013; Costa et al. 2022; Rodriguez et al. 2023) (m, n, and o) *Diagrams were prepared based on descriptions by Melià et al. 2013, photographs by Costa et al. 2022 and Rodriguez et al. 2023. m Pelvis with mild involvement of gluteus medius, and gluteus minimus. n Thighs with severe muscle fat replacement of the anterior compartment including vastus lateralis, vastus intermedius, vastus medialis, and rectus femoris; in the medial compartment adductor magnus is severely affected. Semitendinosus and biceps femoris long head present mild involvement; semimembranosus is preserved with mild hypertrophy. o Legs with severe muscle fat replacement of the posterior compartment including gastrocnemius medialis, gastrocnemius lateralis, and soleus. LGMD D3 HNRNPDL (Berardo et al. 2019; Sun et al. 2019; Malfatti et al. 2020; Vicente et al. 2020; Cotta et al. 2023) (p, q, and r) p Pelvis with severe muscle fat replacement of gluteus maximus, moderate involvement of gluteus medius, and mild involvement of gluteus minimus. q Thighs with severe muscle fat replacement of vastus lateralis, vastus intermedius, and vastus medialis, with superficial preservation and hypertrophy of rectus femoris; severe involvement of semitendinosus, moderate involvement of adductor magnus with preservation and hypertrophy of adductor longus; mild involvement of biceps femoris, and semimembranosus. r Legs with severe muscle fat replacement of tibialis anterior, moderate involvement of soleus and extensor group, with relative preservation of gastrocnemius medialis, and gastrocnemius lateralis. LGMD D4 CAPN3 (Vissing et al. 2016; Cerino et al. 2020; González-Mera et al. 2021; Spinazzi et al. 2021) (s, t, and u) s Pelvis with severe muscle fat replacement of the gluteus minimus. t Thigh with severe muscle fat replacement of adductor magnus, and biceps femoris, moderate involvement of semimembranosus, and mild involvement of semitendinosus. u Legs with moderate involvement of gastrocnemius medialis, and mild involvement of soleus. LGMD D5 COL6A1, COL6A2, COL6A3 (Straub et al. 2012; Fu et al. 2016; Suárez et al. 2018) (v, w, and x). v Pelvis with involvement of glutei muscles. w Thighs with involvement of the periphery of vastus lateralis more than the central part called "sandwich sign" or "striped pattern", a central shadow ("target sign") of involvement in rectus femoris, peripheral involvement of adductor magnus, and relative preservation of sartorius and gracilis. x Legs with peripheral involvement between the soleus, and gastrocnemius medialis, called "rim" of fatty infiltration

Fig. 6

Muscle imaging in treatable and potentially treatable diseases with grading (Wattjes et al. 2010) diagrams of LGMD R28 (a, b, and c), Late onset Pompe disease (d, e, and f), Late onset TK2d (g, h, and i), and POLG deficiency (j, k, and l). Muscle MRI of anti-NXP2/anti-PL7 subacute dermatomyositis (m, n, o, p, q, and r). Muscle MRI of anti-Mi-2 beta chronic dermatomyositis (s, t, u, v, w, and x) of the pelvis (a, d, g, j, m, p, s, and v), thighs (b, e, h, k, n, q, t, and w), and legs (c, f, i, l, o, r, u, and x). LGMD R28 HMGCR treatable with mevalonate (Morales-Rosado et al. 2023 and Yogev et al. 2023). (a, b, and c) *Diagrams were prepared based on photographs of the thighs and legs by Morales-Rosado et al. 2023 and Yogev et al. 2023b Thighs with almost complete muscle fat replacement of all compartments. c Legs with severe muscle fat replacement of gastrocnemius medialis, gastrocnemius lateralis, and soleus. Late onset Pompe disease treatable with enzyme replacement (d, e, and f) (Díaz‐Manera et al. 2021) d Pelvis with severe muscle fat replacement of gluteus medius, and gluteus minimus, with moderate involvement of gluteus maximus. e Thighs with severe muscle fat replacement of adductor magnus and biceps femoris long head, moderate involvement of vastus intermedius, semitendinosus, and semimembranosus, with mild involvement of vastus lateralis, and vastus medialis. f Legs with almost invariable complete preservation of all compartments that in some patients may present mild involvement of gastrocnemius medialis, and soleus. Late onset TK2 deficiency (TK2d) potentially treatable with thymidine and deoxycytidine nucleosides that frequently presents a peculiar combination of facial (with or without ptosis and/or ophthalmoparesis), cervical and axial weakness, with early respiratory insufficiency (g, h, and i) (Domínguez-González et al. 2022). g Pelvis with severe muscle fat replacement of gluteus maximus. h Thighs with severe muscle fat replacement of sartorius and diffuse moth eaten pattern (hachurated graticule) of moderate muscle fat replacement involving gracilis, biceps femoris long head, semitendinosus, and semimembranosus. i Legs with severe muscle fat replacement of gastrocnemius medialis. POLG deficiency (j, k, and l) is nowadays nontreatable but it is potentially treatable with deoxyribonucleoside (Pitceathly et al. 2013; Tasca et al. 2017; Kristiansen et al. 2023). Diagrams based on photographs by Pitceathly et al. 2013, and Tasca et al. 2017. j Pelvis with preservation of glutei muscles. k Thighs with concentric involvement characterized by severe muscle fat replacement from the outer, subcutaneous layers and progresses to the inner profound layers moving distally; there is severe involvement of vastus lateralis, rectus femoris, outer layer of lateral part of vastus intermedius, sartorius, gracilis, outer layer of biceps femoris, and outer layer of semitendinosus with preservation of adductor magnus, vastus medialis, and semimembranosus. l Legs with concentric involvement of the more superficial muscles and preservation of the profound muscles with severe muscle fat replacement of tibialis anterior, extensor group, peroneal group, gastrocnemius medialis, and gastrocnemius lateralis, with preservation of tibialis posterior, and proximal soleus (not shown). Muscle MRI of subacute anti-NXP2/anti-PL7 dermatomyositis of a 25 years old patient with rapid progression of myalgia, weakness, and dyspnea with proximal lower and upper limb weakness, 16.9 times increased creatine kinase (total creatine kinase of 3,210 IU/L, reference < 190 IU/L) T1 (m, n, and o), STIR (p, q, and r). Pelvis (m), thighs (n), and legs (o) with preserved aspect on T1-weighted sequences and diffuse increased signal of muscles and subcutaneous tissue on STIR-weighted sequences of the Pelvis (p), thighs (q), and legs (r) with pronounced enhancement of tibialis posterior. Muscle MRI of chronic anti-Mi-2 beta dermatomyositis of a female patient admitted at 52 years old with a history of proximal lower limbs muscle weakness since 41 years of age, partial improvement with corticosteroid treatment, and 3.2 times increased creatine kinase (total creatine kinase of 529 IU/L, reference < 165 UI/L) T1 (s, t, and u) and STIR (v, w, and x) s Pelvis with moderate involvement of gluteus maximus, gluteus medius, and gluteus mininmus. t Thighs with severe muscle fat replacement of biceps femoris long head, and semimembranosus, and mild involvement of adductor magnus. u Legs with severe muscle fat replacement of soleus. Normal aspect of the muscles on STIR-weighted sequence (v, w, and x). Late onset TK2d: Late onset TK2 deficiency

Fig. 7

Algorithm for imaging differential diagnosis of LGMD based on published articles (Aivazoglou L et al. 2022; Argente‐Escrig et al., 2021; Baranello et al. 2015; Belaya et al. 2015; Berardo et al. 2019; Bohlega et al. 2018; Cerino et al. 2020; Chandrasekharan et al. 2021; Chompoopong and Milone 2023; Cirak et al. 2013; Clement et al. 2008; Coppens et al. 2021; Costa et al. 2022; Cotta et al. 2023; Cotta et al., unpublished data; Díaz‐Manera et al., 2021; Domínguez-González et al. 2022; Dong et al. 2015; Endo et al. 2015; Fu et al. 2016; González-Mera et al. 2021; Haberlova et al. 2014; Hafner et al. 2014; Harris et al. 2017a; Harris et al. 2017b; Hicks et al. 2011; Jonson et al. 2018; Johnson et al. 2019; Kim et al. 2018; Kojima et al. 2017; Kristiansen et al. 2023; Larson et al. 2018; Liang et al. 2015; Llansó et al. 2023; Malfatti et al. 2020; Melià et al. 2013; Meyer et al. 2022; Morales-Rosado et al. 2023; Mroczek et al. 2020; Munot et al. 2022; Nam et al. 2015; Palmio et al. 2015; Panicucci et al. 2023; Pitceathly et al. 2013; Riisager et al.. 2013; Rodriguez et al. 2023; Sandell et al. 2010; Sandell et al. 2013; Savarese et al. 2020; Servián-Morilla et al. 2016; Servián-Morilla et al. 2020; Silva et al. 2019; Spinazzi et al. 2021; Straub et al. 2012; Suárez et al. 2018; Sun et al. 2019; Swan et al. 2023; Tasca et al. 2013; Tasca et al. 2017; Tasca et al. 2018; Tsai et al. 2017; Vicente et al. 2020; Villar Quiles et al. 2020; Vissing et al. 2016; Vissing et al. 2019; Yang et al. 2021; Yogev et al. 2023; Wattjes et al. 2010; Zima et al. 2020). Axial T1-weighted muscle imaging of the pelvis (a, d, g, and j), thighs (b, e, h, and k), and legs (c, f, i, and l). LGMDR1 32 years old female patient (a, b, and c) with the c.2306GA p.(Arg769Gln) homozygous variant in exon 22 of the CAPN3 gene with sandwich sign in vastus lateralis (yellow arrow in b) and target sign in rectus femoris (green arrow in b). LGMD R1 23 years old male patient (d, e, and f) with two heterozygous variants c.328CT (p.Arg110) in exon 2 and c.2306GA (p.Arg769Gln) in exon 22 of the CAPN3 gene with predominant posterior (yellow arrow in e) involvement, compared to the anterior (green arrow in e) of the thighs. LGMD R5 28 years old male patient (g, h, and i) with the homozygous c.525delT, p.(Phe175Leufs20) variant in exon 6 of the gamma-sarcoglycan (SGCG) gene with anterior (green arrow in h) and posterior (yellow arrow in h) involvement of the thighs. LGMD D5 10 years old male patient (j, k, and l) with the homozygous c.911_912delGC p.Gly304Valfs146Ter variant in exon 8 of the COL6A2 gene with slightly more anterior (green arrow in k) than posterior (yellow arrow in k) involvement of the thighs

Pathologists may be aware of the particular radiophenotypes that may help in the interpretation of variants of unknown significance, doubtful immunohistochemical, or Western blot results. Some imaging patterns are well established with a great number of patients, such as those patterns for the most prevalent LGMD (Fig. 2) (Straub et al. 2012, 2018; Winckler et al. 2019; Benarroch et al. 2024) (Table 3) (Bevilacqua et al. 2020; Chakravorty et al. 2020; Cotta et al. 2017; Fanin et al. 2009; Gomez-Diaz et al., 2012; Guglieri et al. 2008; Lo et al. 2008; Lorenzoni et al. 2023; Liu et al. 2019; Norwood et al. 2009; Winckler et al. 2019). LGMD R1 (former LGMD2A), LGMD R2 (former LGMD2B), LGMD R3 (former LGMD2D), LGMD R4 (former LGMD2E), LGMD R5 (former LGMD2C), LGMD R6 (former LGMD2F), LGMD R9 (former LGMD2I), LGMD R22/ LGMD D5 (Bethlem myopathy), and LGMD R23 (merosinopathy) present characteristic imaging patterns. For the most rare LGMD only case reports with muscle imaging that have been published. Their muscular involvement pattern have been here transformed in didactic diagrams for differential diagnosis (Figs. 3, 4, 5, and 6).

An algorithm for imaging differential diagnosis of LGMD was developed based on published articles, and the predominant muscle involvement pattern is used for evaluation (Fig. 7).

Clinical-radiological-pathological approach for the diagnosis of Limb Girdle Muscular Dystrophy

Western blot techniques may be necessary to confirm a deficiency in calpain (LGMD R1, former LGMD2A) (Fig. 8). Immunohistochemical studies have not achieved good reproducibility due to the rapid degradation of the enzyme calpain in the sarcoplasm.

Fig. 8

Limb Girdle Muscular Dystrophy R1, LGMD R1, calpainopathy, muscle imaging, Western blot and muscle biopsy light and electron microscopy findings of a 36 years old male patient with two heterozygous variants in the CAPN3 gene c.865 C > T: p.(Arg289Trp) probably pathogenic and c.619_620delinsGc:p.(Lys207Ala) of uncertain pathogenicity and proof of pathogenicity with muscle biopsy Western blot. a, b, and c. T1-weighted axial Magnetic resonance imaging of the pelvis (a), thighs (b), and legs (c). a. Pelvis with severe muscle fat replacement of gluteus maximus, gluteus medius, and gluteus minimus. b Thighs with muscle fat replacement of adductor magnus (red asterisk * in b), quadriceps femoris, i.e., vastus lateralis, vastus intermedius, rectus femoris, and vastus medialis, with peripheral muscle fat replacement of the vastus lateralis in a sandwich pattern (yellow arrow in b), severe involvement of semimembranosus, semitendinosus, and biceps femoris long head with preservation of biceps femoris short head (green arrow in b), sartorius (yellow asterisk * in b) and gracilis (blue asterisk * in b). c Legs with severe muscle fat replacment of soleus and gastrocnemius medialis (yellow arrow in c), with partial preservation of gastrocnemius lateralis (green arrow in c). STIR-weighted axial Magnetic resonance imaging of the pelvis (d), thighs (e), and legs (f). Thighs with increased signal in left sartorius (yellow arrow in e). Legs with increased signal in tibialis anterior (yellow arrow in f), extensor groups, peroneous groups, and soleus. Western blot with calpain (g and h) and dysferlin (i and j) antibodies of control (g and i) and patient (h and j) with complete negativity of calpain expression in the patient (yellow arrow in h), compared to the control (g) with normal expression of dysferlin in the patient (yellow arrow in j) and in control (i). Right tibialis anterior muscle biopsy (k, l, m, n, o, p, q, r, s, t, u, and v) presenting muscle fibres with rimmed vacuoles (arrows in k and l), necrosis and phagocytosis (arrow in m), myofibre splitting (arrow in n), subsarcolemmal mitochondrial accumulation (arrow in o), oxidative irregularities in moth eaten pattern (arrows in p and q), increased acid phosphatase activity (arrow in r), focal areas of decreased intensity of dysferlin sarcolemmal membrane reaction (black arrows in s) in the same non-necrotic muscle fibres (*) analyzed in serial sections for spectrin (black arrow in t). Sarcolemmal membrane irregularities and subsarcolemmal mitochondrial accumulation (arrow in u) characteristic of lobulated fibres could be observed as well as irregular nucleus with indentations (arrow in v). (a, b, and c T1-weighted magnetic resonance imaging, d, e, and f STIR-weighted magnetic resonance imaging, g and h Western blot with calpain antibodies, i and j Western blot with dysferlin antibodies, k. HE 100x, l. HE 400b, m. Modified Gomori Trichrome 200x, n. ATPase pH 4.6 100x, o. SDH 100x, p. COX-SDH 100x, q. NADH 100x, r. Acid phosphatase 100x, s. Immunohistochemistry anti-dysferlin antibody 400x, t. Immunohistochemistry anti-spectrin antibody 400x, Transmission Electron Microscopy u. 2,500x, and v. 8,000x)

Antibodies directed to proteins of the sarcolemma such as dysferlin (LGMD R2, former LGMD2B) (Fig. 9), and the proteins of the sarcoglycan complex, alpha, beta, gamma, and delta, respectively LGMD R3 (former LGMD2D) (Fig. 10), LGMD R4, LGMD R5, and LGMD R6 present reaction in the membrane. The deficiencies may be noted as decreased intensity of the reaction compared to the intensity in the normal muscle control or areas of gaps in the membrane reaction in some fibres. It is important to evaluate non-necrotic muscle fibres in serial sections with the sarcolemmal protein spectrin to prove membrane integrity, and selective protein deficiency (Fig. 10).

Fig. 9

Limb Girdle Muscular Dystrophy R2, LGMD R2 dysferlinopathy muscle imaging, muscle biopsy light and electron microscopy findings of a 17 years old male patient with two heterozygous variants in the DYSF gene c.5979dupA (p.Glu1994ArgfsTer3), in exon 53 pathogenic and c.619_620delinsGc:p.(Lys207Ala) missense c.1253C > G (p.Pro418Arg), in exon 13 of uncertain pathogenicity with subacute onset clinically simulating inflammatory myopathy and proof of pathogenicity with muscle biopsy immunohistochemistry. a, b, and c T1-weighted magnetic resonance imaging. a. Pelvis with severe muscle fat replacement of gluteus medius, and gluteus minimus. b. Thighs with severe muscle fat replacement and atrophy of the posterior compartment, with severe muscle fat replacement and atrophy of biceps femoris long head (blue arrow), atrophy of semitendinosus, and semimebranosus; moderate involvement of vastus lateralis, vastus intermedius, rectus femoris, and vastus medialis with preservation and hypertrophy of sartorius, and gracilis. c Legs with severe muscle fat replacement and atrophy of soleus (yellow arrow), atrophy of gastrocnemius medialis, and gastrocnemius lateralis, and moderate involvement of peroneus group. Right quadriceps femoris muscle biopsy (d, e, f, g, h, i, j, k, l, m, n, o, p, q, r, s, t, and u) with variation in fibre calibre, atrophy, hypertrophy, endomysial fibrosis and edema (yellow arrow in d and e), necrosis and phagocytosis (inset in e and arrow in f, inset in e is the same field as f), endomysial mononuclear lympho-histiocytic inflammatory infiltrate (blue arrow in e, yellow arrow in g) involving and infiltrating individual non-necrotic muscle fibres (inset in g and arrow in h, inset in g is the same field in h) and subsarcolemmal vacuoles (arrow in i). j Fibre splitting (arrow in j), atrophic type 2B fibres (arrow in k), necrotic fibres (yellow arrow in l and blue arrow in m), intermyofibrillar network accumulation (yellow arrow in m, yellow arrow in o), and increased lysosomal activity (arrow in n). Immunohistochemistry with partial deficiency of sarcolemmal dysferlin expression (asterisks * in p and arrows in r) in non-necrotic muscle fibres with normal reaction for spectrin in serial sections of the same fibres (asterisks * in q and arrows in s). t. Autophagic vacuoles (arrow in t) and inflammatory infiltrate in necrotic fibre. u. Irregular muscle membrane in atrophic fibre (yellow arrow in u) and membrane remnants (blue arrow in u). (a., b., and c. Axial T1-weighted magnetic resonance imaging of the pelvis (a), thighs (b), and legs (c), d. HE 100x, e. HE 100x, f. HE 400x, g. HE 200x, h. HE 400x, i. HE 400x, j. Modified Gomori trichrome 200x, k. ATPase pH4.6 100x, l. SDH 200x, m. COX-SDH 200x, n. Acid phosphatase 100x, o. NADH 200x, p. Immunohistochemistry anti-dysferlin, patient 200x (inset Immunohistochemistry anti-dysferlin, control 200x), q. Immunohistochemistry anti-spectrin, patient 200x (inset Immunohistochemistry anti-spectrin, control 200x), r. Immunohistochemistry anti-dysferlin, patient 400x, s. Immunohistochemistry anti-spectrin, patient 400x, Transmission electron microscopy t. 2,000x, and u. 10,000x)

Fig. 10

Limb Girdle Muscular Dystrophy R3, LGMD R3 alpha-sarcoglycanopathy muscle imaging, muscle biopsy light and electron microscopy findings of a 31 years old female patient with two heterozygous variants in the SGCA gene c.724G > T and c.850 C > T classified as probably pathogenic with proof of pathogenicity through muscle biopsy immunohistochemistry. a, b, and c T1-weighted magnetic resonance imaging. a. Pelvis with severe muscle fat replacement of gluteus maximus (green arrow in a), gluteus medius, and gluteus minimus. b Thigh with severe muscle fat replacement of vastus intermedius, and adductor magnus with relative preservation of the semitendinosus (blue arrow in b), sartorius, and gracilis. c Legs with relative preservation of soleus, gastrocnemius medialis, and gastrocnemius lateralis (yellow arrow in c). Right triceps brachialis muscle biopsy with muscle fat replacement (blue arrow in d) and fibrosis (yellow arrow in d), necrosis with phagocytosis (arrow in e, g, h, j and r), regeneration with basophilic sarcoplasm and increased internalized nuclei (inset in f and arrow in i), slight intrasarcoplasmic deposits of PAS-reactive substances, i.e. glycogen, neutral mucopolysacarides, glycoproteins, mucoproteins, glycoproteins, glycolipids, some insaturated lipids and phopholipids (arrow in k), moderate intrasarcoplasmic lipid deposits (arrow in l), normal checkerboard pattern with type 1 (yellow * in m) and type 2 (black * in m) fibres, subtle irregular areas of slight reduction in oxidative reactions (arrows in n and o), subtle areas of subsarcolemmal accumulation of the intermyofibrillary network (arrows in p and q), increased lysosomal activity and phagocytosis (arrow in r), fibre degeneration (arrow in s), non-necrotic fibres with decreased intensity of the reaction for alpha-sarcoglycan (asterisks * and arrow in t) with integrity of the sarcolemmal membrane demonstrated by serial sections of the same fibres with spectrin reaction (asterisks * and arrow in u). (a., b., and c. Axial T1-weighted magnetic resonance imaging of the pelvis, thighs, and legs, d. HE 100x, e. HE 100x, f. HE 100x, g. HE 200x, h. HE 200x, i. HE 400x, j. Modified Gomori Trichrome 200x, k. PAS 200x, l. Oil-red-O 400x, m. ATPase pH4.6 100x, n. SDH 200x, o. SDH 200x, p. COX-SDH 100x, q. NADH 200x, r. Acid phosphatase 400x, s. Nonspecific esterase 200x, t. Immunohistochemistry anti-alpha-sarcoglycan 200x, u. Immunohistochemistry anti-spectrin 200x)

Proteins of the extracellular matrix may be evaluated in a membranous pattern similar to the membrane pattern. Antibodies are available for alpha-2 laminin (merosin), and alpha-dystroglycan. These proteins may be decreased in the group of Muscular Dystrophy Dystroglycanopathies (MDDG): LGMD R9 (former LGMD2I) (Fig. 11), LGMD R11, LGMD R13, LGMD R14, LGMD R15, LGMD R16, LGMD R19, LGMD R20, and LGMD R24. In theses cases, proof of extracellular membrane integrity may be achieved with the use of laminin gamma-1 or, in some cases, beta-dystroglycan antibodies.

Fig. 11

Limb Girdle Muscular Dystrophy R9, LGMD R9, Fukutin-related proteinopathy or FKRP-pathy muscle imaging, muscle biopsy light and electron microscopy findings of an 11 years old female patient with two heterozygous variants in the FKRP gene, c.826 C > A (p.Leu276Ile) and c.1384 C > T (p.Pro462Ser) with subacute onset, and rapid clinical deterioration simulating autoimmune myositis with characteristic FKRP radiophenotype, and immunohistochemical pattern of alpha-dystroglycanopathy. a, b, and c Axial T1-weighted magnetic resonance imaging of the pelvis (a), thighs (b), and legs (c). a. Pelvis with severe muscle fat replacement of the gluteus minimus (green asterisk *). b. Thighs with severe muscle fat replacement of adductor magnus (yellow asterisk *) and biceps femoris long head (blue asterisk *). Light microscopy of right quadriceps femoris muscle biopsy (b, c, d, e, f, g, h, i, j, k, l, m, n, o, p, q, r, s, t, and u). d. Muscle with variation in fibre calibre, atrophy, hypertrophy, foci of perivascular lymphocytic inflammatory infiltrate (yellow arrows in d, g, and h) with edema (green arrows in d, f, and h), regenerating fibres characterized by basophilic or amphophilic sarcoplasm (arrows in e, i, and k), degeneration and phagocytosis (arrow in j). l. Small groups of type 1 fibres (yellow asterisk * in l) amidst with type 2 fibres (black asterisk * in l). Irregular distribution of the intermiofibrillary network with increased (arrow in m) or decreased (arrow in n) oxidative reaction. Small fibres (arrow in o) with increased lysosomal activity (arrow in p). Normal pattern of Major Histocompatibility Complex I (MHC-I) reation negative in the sarcolemma (arrow in q) with positive vessels internal control with similar reaction in the patient and in the control. Serial sections of the same fibres (asterisks * in r, s, t, and u), with reduction in sarcolemmal reaction of alpha-2 laminin (merosin) and integrity of the sarcolemmal membrane demonstrated by spectrin reaction. (a., b., and c. Axial T1-weighted magnetic resonance imaging of the pelvis, thighs, and legs, d. HE 100x, e. HE 200x, f. HE 100x, g. HE 400x, h. HE 400x, i. HE 400x, j. Modified Gomori Trichrome 400x, k. Modified Gomori Trichrome 400x, l. ATPase pH4.6 100x, m. SDH 200x, n. COX 100x, o. NADH 200x, p. Acid phosphatase 200x, q. Immunohistochemistry anti-MHC-I 200x (patient), inset Immunohistochemistry anti-MHC-I 200x (control), r. Immunohistochemistry anti-alpha-2 laminin (merosin) 200x (patient), s. Immunohistochemistry anti-spectrin 200x (patient), t. Immunohistochemistry anti-alpha-2 laminin (merosin) 400x (patient), u. Immunohistochemistry anti-spectrin 400x (patient)

For the diagnosis of telethonin deficiency, LGMD R7 (former LGMD2G), the antibodies are directed to the sarcomere with positivity inside the sarcoplasm (cytoplasm of the muscle fibre) (Cotta et al. 2024a). The expression of myotilin may be used to indicate sarcomere reaction.

Use of muscle biopsy for Limb Girdle Muscular Dystrophy phenotypic characterization

A list of morphologic light and electron microscopy findings, immunohistochemistry profiles, and Western blot patterns that may be useful for phenotypic characterization in cases of doubtful pathogenicity with variants of unknown significance on molecular studies is provided in Table 5 (Baranello et al. 2015; Barresi et al. 2015; Belaya et al. 2015; Berardo et al. 2019; Boito et al. 2007; Bonnemann 2011; Borg et al. 2009; Brun et al. 2018; Cenacchi et al. 2013; Cerino et al. 2020; Chen et al. 2021; Chompoong and Milone 2023; Christiansen et al. 2022; Cirak et al. 2013; Clement et al. 2008; Coppens et al. 2021; Cossée et al. 2009; Costa et al. 2022; Cotta et al. 2014a, b; Cotta et al. 2021; Cox et al. 2019; Dai et al. 2019; Darin et al. 2007; De Cid et al. 2015; Dinçer et al. 2003; Dong et al. 2015; Endo et al. 2015; Evilä et al. 2014; Fanin M et al., 2003; Fernández-Eulate et al. 2020; Fiorillo et al. 2013; Gamez et al. 2001; Guan et al. 2023; Gundesli et al. 2010; Haberlova et al. 2014; Hafner et al. 2014; Hara et al. 2011; Harris et al. 2017a; Harris et al. 2017b; Ishikawa et al. 2004; Jarry et al. 2007; Johnson et al. 2019; Justel et al. 2023; Klinge et al. 2008; Koss-Harnes et al. 2004; Larson et al. 2018; Malfatti and Richard 2020; Melià et al. 2013; Meyer et al. 2022; Moreira et al. 2000; Morales-Rosado et al. 2023; Mroczek et al. 2020; Munot et al. 2022; Niiyama et al. 2002; Niiyama et al. 2003; Paim et al. 2013; Panicucci et al. 2023; Poppe et al. 2003; Rajakulendran et al. 2011; Riisager et al. 2013; Sabatelli et al. 2003; Sandell et al. 2016; Savarese et al. 2020; Schoser et al. 2005; Selcen et al. 2001; Servián-Morilla et al. 2016; Servián-Morilla et al. 2020; Soontrapa and Liewluck 2022; Starling et al. 2004; Swan et al. 2023; Tasca et al. 2013; Vainzof et al. 2021; van Tol et al. 2019; Vieira et al. 2014; Vihola et al. 2018; Villar Quiles et al. 2020; Vissing et al. 2019; Willis et al. 2022; Zanoteli et al. 2020; Zhang et al. 2022).

Table 5 Useful muscle biopsy findings for Limb Girdle Muscular Dystrophy phenotypic characterization in cases of variants of uncertain significance (Baranello et al. 2015; Barresi et al. 2015; Belaya et al. 2015; Berardo et al. 2019; Boito et al. 2007; Bonnemann 2011; Borg et al. 2009; Brun et al. 2018; Cenacchi et al. 2013; Cerino et al. 2020; Chen et al. 2021; Chompoong and Milone 2023; Christiansen et al. 2022; Cirak et al. 2013; Clement et al. 2008; Coppens et al. 2021; Cossée et al. 2009; Costa et al. 2022; Cotta et al. 2014a; Cotta et al. 2014b; Cotta et al. 2021; Cox et al. 2019; Dai et al. 2019; Darin et al. 2007; De Cid et al. 2015; Dinçer et al. 2003; Dong et al. 2015; Endo et al. 2015; Evilä et al. 2014; Fanin M et al., 2003; Fernández-Eulate et al. 2020; Fiorillo et al. 2013; Gamez et al. 2001; Guan et al. 2023; Gundesli et al. 2010; Haberlova et al. 2014; Hafner et al. 2014; Hara et al. 2011 Harris et al. 2017a, Harris et al. 2017b; Ishikawa et al. 2004; Jarry et al. 2007; Johnson et al. 2019; Justel et al. 2023; Klinge et al. 2008; Koss-Harnes et al. 2004; Larson et al. 2018; Malfatti and Richard 2020; Melià et al. 2013; Moreira et al. 2000; Munot et al. 2022; Meyer et al. 2022; Morales-Rosado et al. 2023; Mroczek et al. 2020; Niiyama et al. 2002; Niiyama et al. 2003; Paim et al. 2013; Panicucci et al. 2023; Poppe et al. 2003; Rajakulendran et al. 2011; Riisager et al. 2013; Sabatelli et al. 2003; Sandell et al. 2016; Savarese et al. 2020; Schoser et al. 2005; Selcen et al. 2001; Servián-Morilla et al. 2016; Servián-Morilla et al. 2020; Soontrapa and Liewluck 2022; Starling et al. 2004; Swan et al. 2023; Tasca et al. 2013; Vainzof et al. 2021; van Tol et al. 2019; Vieira et al. 2014; Vihola et al. 2018; Villar Quiles et al. 2020; Vissing et al. 2019; Willis et al. 2022; Zanoteli et al. 2020; Zhang et al. 2022)

Conclusion

The differential diagnosis of the most common Limb Girdle Muscular Dystrophy subtypes is possible with the analysis of data from the anamnesis, clinical examination, and results of the laboratorial, neurophysiological, imaging, muscle biopsy, and molecular pathology findings.

After the adequate phenotypic characterization, the diagnosis is confirmed by molecular pathology studies. If molecular studies are inconclusive, muscle imaging may provide a radiophenotype that may guide the diagnostic investigation.

Muscle biopsy plays an important role in the molecular era. It may provide functional proof of pathogenicity for variants of unknown significance by the identification of deficiencies in the expression of specific proteins (gene products). Immunohistochemistry, immunofluorescence, and Western blot may identify the deficiency of proteins of the most common Limb Girdle Muscular Dystrophy subtypes, such as sarcoglycans (alpha, beta, gamma, and delta), dysferlin, telethonin, alpha-2-laminin or merosin (partial merosin deficiency in Limb Girdle Muscular Dystrophy opposed to complete merosin deficiency in congenital muscular dystrophy that starts symptoms in the first two years of age), alpha-dystroglycan, and calpain (Western blot) that are commercially available. Muscle biopsy is also important for the differential diagnosis between treatable autoimmune myositis (that may present clinical improvement with immunosuppressants), and hereditary muscular dystrophies.