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)

LGMD R1/ CAPN3

Calpain 3

Muscle biopsy may present dystrophic features (muscle fat replacement, endomysial fibrosis, atrophy, hypertrophy, fibre splitting, necrosis, phagocytosis, and regeneration) if the muscle site chosen corresponds to a clinically weak muscle. Vastus lateralis muscle biopsy may be normal in early stages of the disease specially if muscle imaging demonstrates normal aspect. There may be lobulated or trabeculated fibres, characterized by reduced diameter with subsarcolemmal, and intermyofibrillary mitochondrial proliferation and irregular sarcolemmal membrane (Cotta et al. 2014a). Anti-calpain-3 antibodies are commercially available for muscle biopsy Western blot. The results may be of either complete absence of the calpain band or partial reduction on its intensity. False negatives may be observed in circa 20 to 30% of calpainopathy patients with normal Western blot calpain-3 bands (Malfatti and Richard 2020). False positives with secondary reduction in the intensity of the calpain-3 band in Western blot in patients with dysferlinopathy (LGMD R2), FKRP-pathy (LGMD R9), titinopathy (LGMD R10), and anoctaminopathy (LGMD R12) (Cotta et al. 2021; Malfatti and Richard 2020). Transmission electron microscopy may demonstrate disruption of the sarcomeric structure associated with myofibrillar disorganization, fragmented nuclei with focally detached

chromatin, and increased subsarcolemmal glycogen deposits (Fanin M et al., 2003)

LGMD R2/ DYSF

Dysferlin

Muscle biopsy usually presents dystrophic features with necrosis, phagocytosis, and regeneration. Foci of perivascular lymphocytic inflammatory infiltrate are common (Cotta et al. 2014a). Anti-dysferlin antibodies are commercially available for immunohistochemistry, immunofluorescence, and Western blot in muscle biopsy tissue. The results may be complete absence of dysferlin reaction in the sarcolemmal membrane or partial reduction in its intensity in some fibres. Immunohistochemistry for use in monocytes in peripheral blood smears were published (Cox et al. 2019) but in our experience, in patients with partial dysferlin deficiency, the results are much more difficult to interpret compared to muscle biopsy tissue. False positives with secondary partial dysferlin deficiency were described in calpainopathy (LGMD R1), caveolinopathy (gene CAV3), dystrophinopathy (gene DMD), sarcoglycanopathy (LGMD R3, LGMD R4, LGMD R5, and LGMD R6), anoctaminopathy (LGMD R12), GNE myopathy (GNE gene), and myotilinopathy (MYOT gene) (Cotta et al. 2021). Transmission electron microscopy may demonstrate small defects in the sarcolemmal membrane from 0.11 to 1.8 µm sometimes associated with thickened basal lamina over the defects, replacement of the plasma membrane by one to multiple layers of small vesicles, papillary projections with disintegration of fibres, small subsarcolemmal vacuoles undergoing exocytosis, and subsarcolemmal regions with rough endoplasmic reticulum and abundant free ribosomes (Selcen et al. 2001)

LGMD R3/ SGCA

Alpha sarcoglycan

Muscle biopsy usually presents dystrophic pattern with muscle fat replacement, endomysial fibrosis, atrophy, hypertrophy, fibre splitting, necrosis, phagocytosis, and regeneration (Cotta et al. 2014a). Anti-alpha-sarcoglycan antibodies are commercially available for immunohistochemistry, immunofluorescence, and Western blot (Vainzof et al. 2021; Fernández-Eulate et al. 2020). The results may be either complete deficiency in sarcolemmal expression or partial reduction in the intensity of the reaction in some fibres. Pathogenic variants in the alpha-sarcoglycan gene (SGCA) may cause a secondary reduction in the expression of any sarcoglycan subunit (Klinge et al. 2008; Vainzof et al. 2021). Therefore the deficiency of one protein of the sarcoglycan complex predicts a pathogenic variant in any of the four sarcoglycan genes (SGCA, SGCB, SGCG, or SGCD)

LGMD R4/ SGCB

Beta sarcoglycan

Muscle biopsy usually presents dystrophic pattern with muscle fat replacement, endomysial fibrosis, atrophy, hypertrophy, fibre splitting, necrosis, phagocytosis, and regeneration (Cotta et al. 2014a). Anti-beta-sarcoglycan antibodies are commercially available for immunohistochemistry, immunofluorescence, and Western blot (Vainzof et al. 2021; Fernández-Eulate et al. 2020). The results may be either complete deficiency in sarcolemmal expression or partial reduction in the intensity of the reaction in some fibres. Pathogenic variants in the beta-sarcoglycan gene (SGCB) may cause a secondary reduction in the expression of any sarcoglycan subunit (Klinge et al. 2008; Vainzof et al. 2021). Therefore the deficiency of one protein of the sarcoglycan complex predicts a pathogenic variant in any of the four sarcoglycan genes (SGCA, SGCB, SGCG, or SGCD)

LGMD R5/ SGCG

Gamma sarcoglycan

Muscle biopsy usually presents dystrophic pattern with muscle fat replacement, endomysial fibrosis, atrophy, hypertrophy, fibre splitting, necrosis, phagocytosis, and regeneration (Cotta et al. 2014a). Anti-gamma-sarcoglycan antibodies are commercially available for immunohistochemistry, immunofluorescence, and Western blot (Vainzof et al. 2021; Fernández-Eulate et al. 2020). The results may be either complete deficiency in sarcolemmal expression or partial reduction in the intensity of the reaction in some fibres. Pathogenic variants in the gamma-sarcoglycan gene (SGG) may cause a secondary reduction in the expression of any sarcoglycan subunit (Klinge et al. 2008; Vainzof et al. 2021). Therefore the deficiency of one protein of the sarcoglycan complex predicts a pathogenic variant in any of the four sarcoglycan genes (SGCA, SGCB, SGCG, or SGCD)

LGMD R6/

SGCD

Delta-sarcoglycan

Muscle biopsy usually presents dystrophic pattern with muscle fat replacement, endomysial fibrosis, atrophy, hypertrophy, fibre splitting, necrosis, phagocytosis, and regeneration (Cotta et al. 2014a). Anti-delta-sarcoglycan antibodies are commercially available for immunohistochemistry, immunofluorescence, and Western blot (Vainzof et al. 2021; Fernández-Eulate et al. 2020). The results may be either complete deficiency in sarcolemmal expression or partial reduction in the intensity of the reaction in some fibres. Pathogenic variants in the delta-sarcoglycan gene (SGCD) may cause a secondary reduction in the expression of any sarcoglycan subunit (Klinge et al. 2008; Vainzof et al. 2021). Therefore the deficiency of one protein of the sarcoglycan complex predicts a pathogenic variant in any of the four sarcoglycan genes (SGCA, SGCB, SGCG, or SGCD)

LGMD R7/ TCAP

Telethonin

Muscle biopsy may present either dystrophic pattern or myopathic abnormalities with variation in fibre diametre, lobulated/ trabeculated fibres, and type 1 fibre atrophy without type 1 fibre predominance (Cotta et al. 2014b; Paim et al. 2013). Anti-telethonin antibodies are commercially available for Western blot, immunohistochemistry, and immunofluorescence (Moreira et al. 2000; Cotta et al. 2014b; Cotta et al. 2024a; Paim et al. 2013). The deficiency is noted as a reduction in the sarcomeric reaction. A complete absence of telethonin reaction may be observed in the sarcoplasm. A partial reduction in the Western band of telethonin was described (Barresi et al. 2015). Transmission electron microscopy may demonstrate endomysial fibrosis, fibre and myofibre splitting, fibre degeneration, intrasarcoplasmic glycogen deposits, and autophagic vacuoles containing sarcoplasmic and membrane debris (Cotta et al. 2014b)

LGMD R8/ TRIM32

Tripartite motif-containing 32

Myopathic abnormalities with variation in fibre calibre, nuclear internalization, oxidative irregularities, fibre splitting or dystrophic features with necrosis, endomysial fibrosis, and rimmed vacuoles (Cossée et al. 2009; Johnson et al. 2019). Western blot studies detected reduction in TRIM32 protein in muscles from patients with the truncating c.1560delC mutation in the TRIM32 gene (Borg et al. 2009) and in experimental studies of cultured cells infected with TRIM32 mutant plasmids (Guan et al. 2023). Transmission electron microscopy may demonstrate from scattered to a myriad of small vacuoles surrounded by membranes inside muscle fibres and some coalescescing membrane-bound spaces, with aspect of sarcotubular myopathy even when light microscopy does not reveal vacuoles (Schoser et al. 2005; Borg et al. 2009)

LGMD R9/ FKRP

Fukutin-related protein

Muscle biopsy in LGMD R9 may present dystrophic pattern with muscle fat replacement, endomysial fibrosis, necrosis, phagocytosis, and regeneration (Cotta et al. 2014a). Perivascular lymphocytic inflammatory infiltrate may be observed (Darin et al. 2007; Cotta et al. 2014a). LGMD R9 is part of the alpha-dystroglycanopathy subgroup of Limb Girdle Muscular Dystrophies. Therefore secondary reductions may be observed in the expression of alpha-dystroglycan, and alpha-2 laminin (merosin) (Cotta et al. 2014a; Poppe et al. 2003; Villar Quiles et al. 2020; Willis et al. 2022). Anti-merosin antibodies are commercially available for immunohistochemistry, immunofluorescence, and Western blot. Merosin is an extracellular protein linked to the sarcolemmal membrane, therefore, muscle biopsy may demonstrate strong membrane merosin reaction. Patients with LGMD R9 may present isolated negative non-necrotic fibres on immunohistochemistry (Poppe et al. 2003; Cotta et al. 2014a). Antibodies anti-alpha-dystroglycan may present variable membrane reaction depending on the clone, and the technique used either immunohistochemistry, Western blot, or immunofluorescence. Transmission electron microscopy may demonstrate abnormalities in the basal lamina that may demonstrate skein-like structure and irregular texture; severe loss of myofilaments, endoplasmic reticulum dilation and proliferation, swollen T tubules, supernumerary junctional sarcoplasmic reticulum components, triad reduplication, and subsarcolemmal lipid droplets associated with mitochondria (Boito et al. 2007)

LGMD R10/ TTN

Titin

Muscle biopsy may demonstrate myopathic changes such as nuclear internalization or a dystrophic pattern, myotilin reactive myofibrillar deposits (Harris et al. 2017b), and rimmed vacuoles (Evilä et al. 2014). The use of antibodies anti-C-terminal fragment of the titin in Western blot in muscle biopsy tissue was reported (Harris et al. 2017b; Savarese et al. 2020). Patients with limb girdle weakness and early joint contractures without cardiomyopathy related to the TTN gene may present ultrastructural changes such as rimmed vacuoles with degradation products, lamellated myeloid structures, subsarcolemmal and intermyofibrillary autophagic material, nemaline bodies in areas of accumulation of thin filaments, organelles, and debris, myofibrillar disorganization, dissolution of the M-band structure with disintegration of myosin filament with intact Z disk, large tubulo-filamentous inclusions disrupting the myofibrillar network, intranuclear inclusions, and cytoplasmic bodies surrounded by amorphous material with cellular debris, and filaments (De Cid et al. 2015)

LGMD R11/ POMT1

Protein-O-mannosyltransferase 1

Muscle biopsy may present fibre type disproportion, and nuclear internalization (Haberlova et al. 2014; Hafner et al. 2014). LGMD R11 is part of the alpha-dystroglycanopathy subgroup of Limb Girdle Muscular Dystrophies. Therefore secondary reductions may be observed in the expression of alpha-dystroglycan (Haberlova et al. 2014; Hafner et al. 2014). Alpha-dystroglycan is an extracellular protein linked to the sarcolemmal membrane, therefore, muscle biopsy may demonstrate strong membrane merosin reaction. Antibodies anti-alpha-dystroglycan may present variable membrane reaction depending on the clone, and the technique used either immunohistochemistry, Western blot, or immunofluorescence. Transmission electron microscopy may demonstrate discontinuities and detachments of the basal lamina from the plasmalemma of the muscle fibres with preservation of the basal lamina from the intramuscular nerves, nuclear abnormalities of the muscle fibres with hypercondensed heterochromatin clumps, complete detachment of peripheral heterochromatin from the nuclear envelope, nuclear envelope with dilated cisterna and marked loss of nuclear pores with preserved nuclear lamina, irregular nuclei with papillary projections of the nuclear envelope lacking heterochromatin, apoptosis with highly condensed, marginated chromatin at the inner surface of the nuclear envelope, dilated endoplasmic reticulum, condensed mitochondria, and satellite cells with condensed chromatin with half-moon-like shape (Sabatelli et al. 2003)

LGMD R12/ ANO5

Anoctamin 5

Muscle biopsy may demonstrate either dystrophic abormalities with necrosis, phagocytosis, and regeneration, or nonspecific myopathic abnormalities such as nuclear internalization, variation in fibre calibre, groups of angulated atrophic fibres simulating neurogenic abnormalities, or mitochondrial abnormalities (Christiansen et al. 2022). Transmission electron microscopy may demonstrate multifocal sarcolemmal lesions, and subsarcolemmal accumulation of vesicles (Soontrapa and Liewluck 2022; Vihola et al. 2018). Antibodies anti-anoctamin 5 clone N421A/85 for Western blot detected reduction in the bands in LGMD R12 patients (Soontrapa and Liewluck 2022; Vihola et al. 2018). The same antibodies did not present satisfatory results with immunohistochemistry, and immunofluorescence (Soontrapa and Liewluck 2022; Vihola et al. 2018). Transmission electron microscopy may demonstrate focal duplication of the muscle basement membrane, increased and disorganized endomysial extracellular matrix, collagen protofibrils spread out haphazardly in a starburst pattern in the endomysium without internal spacing and intermingling with the obscured basement membrane, strands of dense flocculent material close to the basement membrane, and disorganization of the extracellular environment close to the muscle fibre (Jarry et al. 2007)

LGMD R13/ FKTN

Fukutin

Muscle biopsy may demonstrate dystrophic pattern with endomysial fibrosis, variation in fibre calibre, degeneration, and regeneration. In 38% of the patients a lymphocytic inflammatory infiltrate may be observed (Fiorillo et al. 2013; Riisager et al. 2013). LGMD R13 is part of the alpha-dystroglycanopathy subgroup of Limb Girdle Muscular Dystrophies. Therefore secondary reductions may be observed in the expression of the extracellular proteins alpha-dystroglycan, and alpha-2 laminin (merosin) (Fiorillo et al. 2013; Riisager et al. 2013). Anti-merosin antibodies are commercially available for immunohistochemistry, immunofluorescence, and Western blot with strong membrane pattern of reaction. Antibodies anti-alpha-dystroglycan may present variable membrane reaction in immunohistochemistry, Western blot, or immunofluorescence, according to the specific clone used

LGMD R14/ POMT2

Protein-O-mannosyltransferase 2

Muscle biopsy may demonstrate variable presentation from slight myopathic abnormalities to dystrophic pattern. Variation in fibre calibre, nuclear internalization, atrophy, hypertrophy, type 1 fibre predominance, necrosis, regeneration, and endomyosial fibrosis, and muscle fat replacement. About 4% of the patients may present lymphocytic inflammatory infiltrate (Brun et al. 2018; Chen et al. 2021; Panicucci et al. 2023). LGMD R14 is part of the alpha-dystroglycanopathy subgroup of Limb Girdle Muscular Dystrophies. Secondary reductions may be observed in the expression of the extracellular proteins alpha-dystroglycan, and alpha-2 laminin (merosin) that are commercially available (Brun et al. 2018; Chen et al. 2021; Panicucci et al. 2023). Anti-merosin antibodies present strong reaction in the sarcolemmal membrane. Alpha-dystroglycan presents variable results depending on the clone, and the specific technique either immunohistochemistry, immunofluorescence, or Western blot

LGMD R15/ POMGNT1

O-linked mannose beta1,2-N-acetylglucosaminyltransferase

Muscle biopsy may present dystrophic pattern, and alpha-dystroglycan deficiency by immunohistochemistry, immunofluorescence or Western blot (Clement et al. 2008). LGMD R15 is part of the alpha-dystroglycanopathy subgroup of Limb Girdle Muscular Dystrophies. Antibodies anti-alpha-dystroglycan presents variable results depending on the clone, and the technique employed: immunohistochemistry, immunofluorescence, or Western blot

LGMD R16/ DAG1

Dystroglycan 1

Muscle biopsy may present dystrophic pattern, and deficiency in the reaction for alpha-dystroglycan commercially available antibodies by immunohistochemistry, immunofluorescence, or Western blot with variable results (Dai et al. 2019; Dinçer et al. 2003; Dong et al. 2015; Hara et al. 2011). LGMD R16 is part of the alpha-dystroglycanopathy subgroup of Limb Girdle Muscular Dystrophies and antibodies anti-alpha-dystroglycan may detect primary alpha-dystroglycan deficiency

LGMD R17/ PLEC

Plectin

Muscle biopsy may demonstrate myopathic changes with nuclear internalization, angulated atrophic fibres, or dystrophic pattern with endomyosial fibrosis, necrosis, and basophilic fibres. Transmission electron microscopy may demonstrate increased gap between the sarcolemmal membrane and the sarcomere (Gundesli et al. 2010; Mroczek et al. 2020). There is a report on Western blot deficiency of the plectin band with anti-plectin antibodies used in fibroblast culture derived from LGMD R17 patient with epidermolysis bullosa (Koss-Harnes et al. 2004)

LGMD R18/ TRAPPC11

Trafficking protein particle complex 11

Muscle biopsy may demonstrate either myopathic abnormalities without dystrophic features (Justel et al. 2023) or dystrophic pattern (Larson et al. 2018; Munot et al. 2022). Myopathic changes include fibre size variability, nuclear internalization hypercontracted fibres, blurred irregularities in oxidative reactions (SDH), type 1 fibre predominance, and selective type 2 fibre atrophy without dystrophic features such as endomysial fibrosis or muscle fat replacement (Justel et al. 2023; Munot et al. 2022). LGMD R18 is part of the alpha-dystroglycanopathy subgroup of Limb Girdle Muscular Dystrophies. Antibodies IIH6 and VIA4 anti-alpha-dystroglycan may present decreased band in Western blot and decreased sarcolemmal reaction in isolated fibres with integrity of the extracelullar matrix demonstrated by normal sarcolemmal reaction for beta-dystroglycan (Justel et al. 2023; Larson et al. 2018; Munot et al. 2022). Reduction in laminin alpha-2 (merosin) expression may be observed by immunohistochemistry (Munot et al. 2022). Transmission electron microscopy may demonstrate focal Z-band streamings, disruption of the sarcomere in necrotic fibres, and electron-dense degraded material (Justel et al. 2023)

LGMD R19/ GMPPB

GDP-mannose pyrophosphorylase B

Muscle biopsy may demonstrate from myopathic changes to dystrophic pattern. LGMD R19 is part of the alpha-dystroglycanopathy subgroup of Limb Girdle Muscular Dystrophies. Therefore partial reduction in the expression of alpha-dystroglycan may be observed on immunohistochemistry, immunofluorescence, or Western blot (Belaya et al. 2015). Transmission electron microscopy may demonstrate neuromuscular junction abnormalities such as simplification, absence, or degeneration of the post-synaptic folds, dilated vesicles on the junctional sarcoplasm with normal pre-synaptic endings (Chompoong and Milone 2023)

LGMD R20/ CRPPA

Isoprenoid synthase domain containing protein

Muscle biopsy may demonstrate dystrophic pattern with variation in fibre calibre, necrosis, phagocytosis, and regeneration. R20 is part of the alpha-dystroglycanopathy subgroup of Limb Girdle Muscular Dystrophies. Reduced expression of alpha-dystroglycan may be observed by immunohistochemistry, immunofluorescence, or Western blot (Baranello et al. 2015; Cirak et al. 2013; Tasca et al. 2013; van Tol et al. 2019)

LGMD R21/ POGLUT1

Protein O-Glucosyltransferase 1

Muscle biopsy may demonstrate from slight myopathic changes to dystrophic pattern. R21 is part of the alpha-dystroglycanopathy subgroup of Limb Girdle Muscular Dystrophies. Thus, there may be reduced expression of alpha-dystroglycan with variable results by immunohistochemistry, immunofluorescence, or Western blot (Servián-Morilla et al. 2016, 2020)

LGMD R22/ COL6A1, COL6A2, and COL6A3

Alpha 1 type VI collagen, Alpha 2 type VI collagen, and Alpha 3 type VI collagen

Muscle biopsy may demonstrate dystrophic features with endomysial fibrosis, and isolated fibres with necrosis, phagocytosis, and regeneration. Even though antibodies anti-collagen VI are available commercially, the sensitivity is higher for the congenital muscular dystrophy clinical presentation (Ullrich congenital muscular dystrophy) than for the late onset form LGMD R22/ Bethlem myopathy (Bonnemann 2011; Zanoteli et al. 2020). Transmission electron microscopy may demonstrate increased space between muscle fibres and adjacent connective tissue (Ishikawa et al. 2004), irregularities and folds in the plasma membrane and loose connection between the basal lamina, and the extracellular matrix (Lampe and Bushby 2005), muscle basal lamina thickening of 50 to 80 nm and ectopic membranous material external to the basal lamina (Niiyama et al. 2002); capillary abnormalities such as narrowed lumens, fenestration (small discontinuities in endothelium), endothelial cells with enlarged nuclei, increased number of pinocytotic vesicles, dense cytoplasm, and replication of the capillary basement membrane (Niiyama et al. 2003)

LGMD R23/ LAMA2

Laminin alpha 2 chain of merosin

Muscle biopsy may demonstrate dystrophic features. Antibodies anti-merosin may be useful to demonstrate partial deficiency by immunohistochemistry, immunofluorescence, or Western blot. Commercially available anti-merosin antibodies provide consistent results that allow the identification of partial deficiencies (Meyer et al. 2022). Transmission electron microscopy may demonstrate whorled membranous debris, and/or 16–18-nm filaments in a myonucleus (Rajakulendran et al. 2011)

LGMD R24/ POMGNT2

Protein O-linked mannose N-acetylglucosaminyltransferase 2

Muscle biopsy may demonstrate from myopathic to dystrophic changes. Myopathic changes may be nuclear internalization. Dystrophic pattern includes endomysial fibrosis, necrosis, and regeneration. R24 is part of the alpha-dystroglycanopathy subgroup of Limb Girdle Muscular Dystrophies. Antibodies anti-alpha-dystroglycan clone VIA4-1 may demonstrate negative reactions on muscle biopsies with either myopathic or dystrophic changes, detectable by immunohistochemistry, or Western blot, compared to normal reaction for beta-dystroglycan (Endo et al. 2015)

LGMD R25/ BVES

Blood vessel epicardial substance (also called POPDC1)

Muscle biopsy may demonstrate dystrophic pattern with endomysial fibrosis, variation in fibre calibre, atrophy, and hypertrophy (Swan et al. 2023). Experimental studies in animal models have demonstrated the reduction in the expression of the POPDC1 protein by immunofluorescence and Western blot (Swan et al. 2023)

LGMD R26/ POPDC3

Popeye domain-containing protein

Muscle biopsy may demonstrate variation in endomyosial fibrosis, variation in fibre calibre, nuclear internalization, necrosis, whorled fibres, and preserved ultrastructural aspect (Vissing et al. 2019; Zhang et al. 2022). Experimental studies had demonstrated the reduction in the expression of the POPDC3 protein by Western blot (Vissing et al. 2019)

LGMD R27/ JAG2

Jagged 2

Muscle biopsy may demonstrate from myopathic to dystrophic changes, including nuclear internalization, variation in fibre calibre, fibre splitting, lobulated fibres, moth eaten and core-like oxidative irregularities, necrosis, phagocytosis, endomysial fibrosis, and muscle fat replacement (Coppens et al. 2021). An increased number of satellite cells my be revealed by immunostaining for PAX7 and MYF5 compared to control muscle biopsies (Coppens et al. 2021)

LGMD R28/ HMGCR

3-Hydroxy-3-methylglutaryl-CoA reductase

Muscle biopsy may demonstrate degeneration, phagocytosis, prominent fibre size variation, internalized nuclei, and endomysial fibrosis without rimmed vacuoles. Transmission electron microscopy may demonstrate subsarcolemmal mitochondrial clusters, and myelin bodies. Antibodies anti-HMGCR may demonstrate deficiency in the expression of HMGCR (Morales-Rosado et al. 2023)

LGMD D1/ DNAJB6

HSP-40 homologue subfamily B, number 6

Muscle biopsy may demonstrate variation in fibre calibre, nuclear internalization, rimmed vacuoles, dark myofibrillar accumulation on HE that are dark blue on Modified Gomori Trichrome, with positive immunohistochemical reaction for myotilin, alpha–beta-crystalline, desmin, ubiquitin, TDP-43, p62, and SMI-31 (Sandell et al. 2016). Transmission electron microscopy may demonstrate disorganization and widening of the Z disks (Sandell et al. 2016)

LGMD D2/ TNPO3

Transportin 3

Muscle biopsy may demonstrate variation in fibre calibre, diffuse atrophy, increased nuclei with central clear areas, increased acid phosphatase activity, COX negative fibres, and rimmed vacuoles. Immunohistochemistry may demonstrate myofibrilar accumulation in 86% of the cases with abnormal intrasarcoplasmic reaction for desmin, and myotilin (Cenacchi et al. 2013; Costa et al. 2022; Gamez et al. 2001; Melià et al. 2013). Transmission electron microscopy may demonstrate abnormal mitochondriae with paracrystalline inclusions, myelinoid figures, myofibrillar disorganization, autophagic vacuoles, accumulation of electron dense material with density similar to the Z disks, intranuclear, and intrasarcoplasmic non-ramification filamentous inclusions from 18 to 20 nm (Cenacchi et al. 2013; Gamez et al. 2001; Melià et al. 2013). Experimental studies demonstrated abnormal expression of the transportin 3 protein by immunofluorescence with a weak and dispersed pattern with some aggregates (Costa et al. 2022)

LGMD D3/ HNRNPDL

Heterogeneous nuclear ribonucleoprotein D-like

Muscle biopsy may demonstrate variation in fibre calibre, perimysial fibrosis, necrosis, rimmed vacuoles, angulated atrophic fibres, type 2 fibre predominance, groups of type 1, and type 2 fibres. Transmission electron microscopy may demonstrate vacuoles with membranous remnants, myeloid bodies, glycogen granules, cytoplasmic bodies, tubulofilamentous inclusions, and spread filamentous bundles of Z disk material (Berardo et al. 2019; Starling et al. 2004; Vieira et al. 2014)

LGMD D4/ CAPN3

Calpain 3

Muscle biopsy may present reduction in the calpain band by Western blot (Cerino et al. 2020)

LGMD D5/ COL6A1, COL6A2, and COL6A3

Alpha 1 type VI collagen, Alpha 2 type VI collagen, and Alpha 3 type VI collagen

Muscle biopsy may demonstrate dystrophic features with endomysial fibrosis, and isolated fibres with necrosis, phagocytosis, and regeneration. Even though antibodies anti-collagen VI are available commercially, the sensitivity is higher for the congenital muscular dystrophy clinical presentation (Ullrich congenital muscular dystrophy) than for the late onset form LGMD R22/ Bethlem myopathy (Bonnemann 2011; Zanoteli et al. 2020). Transmission electron microscopy may demonstrate increased space between muscle fibres and adjacent connective tissue (Ishikawa et al. 2004), irregularities and folds in the plasma membrane and loose connection between the basal lamina, and the extracellular matrix (Lampe and Bushby 2005), muscle basal lamina thickening of 50 to 80 nm and ectopic membranous material external to the basal lamina (Niiyama et al. 2002); capillary abnormalities such as narrowed lumens, fenestration (small discontinuities in endothelium), endothelial cells with enlarged nuclei, increased number of pinocytotic vesicles, dense cytoplasm, and replication of the capillary basement membrane (Niiyama et al. 2003)