Clinical manifestations

Presentation and course

Limb-girdle dystrophy occurs in both sexes, with onset between the second and sixth decade, usually in late childhood or early adulthood, although onset can occur at almost any age. Weakness in many cases begins in the pelvic girdle musculature (Leyden and Mobius type) and then spreads to the pectoral musculature. However, in the reverse pattern (Erb type), it is not unusual to see the pectoral girdle affected first. In rarer cases, both regions are affected simultaneously. Early facial, distal, or extraocular muscle involvement is not seen. The average interval before symptoms appear in the initially unaffected girdle musculature is about 5 to 10 years, although it can be longer. Autosomal dominant forms of limb-girdle muscular dystrophy tend to occur later in life and progress relatively slowly, although exceptions occur (40; 11; 343; 486; 247). Weakness is progressive, and loss of ambulation occurs earlier when the disease begins in the pelvic girdle, but progression can be slow or rapid. Eventually, virtually all muscles in the body are affected, but the relative distribution of affected and relatively spared muscles determines the characteristic clinical appearance in limb-girdle muscular dystrophy.

Although the severity and time course of weakness in individual muscles may vary, the overall pattern of involvement seems to be similar in pectoral and pelvic forms of limb-girdle muscular dystrophy. The trapezius, serratus anterior, sternal head of pectoralis major, spinati, biceps, and brachioradialis muscles are involved early in the pectoral form. The deltoid muscle may be relatively spared, but this muscle can be difficult to test when the subject is unable to fixate the scapula. The presence of weakness and atrophy in certain muscle groups, with relative sparing of others, often produces a characteristic appearance: drooped shoulders, scapular winging, characteristic anterior axillary fold (due to wasting of the sternal head of the pectoralis major), and "Popeye" arms (due to wasted arm muscles and spared deltoids); however, this pattern is more commonly demonstrated in fascioscapulohumeral muscular dystrophy (FSHD). In the pelvic form of limb-girdle muscular dystrophy, sacrospinalis, quadriceps, hamstrings, and hip muscles are especially involved, causing excessive lumbar lordosis and a waddling gait. Facial muscles are uninvolved in limb-girdle muscular dystrophy until the patient is severely disabled from limb weakness. Why muscles of one region should be selectively targeted remains unknown.

Pseudohypertrophy of calf muscles has been reported in some cases of autosomal recessive limb-girdle muscular dystrophy but is not invariably present (240; 36). Muscle tendon reflexes are preserved in the early stages but are lost as the disease progresses. Ankle jerks often can still be elicited when all other reflexes are lost. Cardiac involvement is seen late in a small minority of patients. Creatine kinase is variably raised (2x to 350x) in recessive cases and is either normal or only mildly increased (to 6x) in dominant patients (64).

The progressive clinical course may eventually lead to loss of ambulation, especially if the pelvic girdle is involved at an early stage. It is often decades before the disease produces severe physical disability and muscle contractures. In terminal stages, patients may have respiratory muscle involvement leading to respiratory failure, pneumonia, and death. Some patients with limb-girdle muscular dystrophy have axial muscle involvement and relatively early respiratory failure (479). The clinical course is variable, however, and some patients have a normal lifespan and may remain ambulatory throughout (40; 11; 343; 486; 91; 403).

Learning difficulties and mild intellectual disability are not rare in limb-girdle muscular dystrophy. Cognitive deficits in limb-girdle muscular dystrophy are thought to occur from defects in assembly and processing of dystroglycans in both muscles and neurons in dystroglycanopathy syndromes (341).

Chronic pain appears to be a prevalent problem in people with limb-girdle muscular dystrophy, with a negative impact on everyday life (532).

Prognosis and complications

Limb-girdle dystrophies, like all muscular dystrophies, are progressive disorders. The rate of progression, however, varies widely in different types of limb-girdle muscular dystrophies and between individuals. Autosomal recessive limb-girdle muscular dystrophy of childhood generally progresses more rapidly, leading to loss of ambulation in late childhood or adolescence and death in early adulthood (36; 563; 149; 14), although milder cases are also seen. Autosomal dominant forms of limb-girdle muscular dystrophy, even those that begin in early childhood, are more benign and life expectancy can be normal (40; 11; 343; 486; 247).

Physical disability in limb-girdle muscular dystrophy is dependent on the distribution and severity of muscle weakness. Not surprisingly, loss of walking ability occurs earlier when the disease begins in the pelvic girdle. The interval between onset of symptoms and inability to walk is highly variable. Muscle contractures may contribute to severe disability in later stages. Respiratory muscle impairment can occur, with muscles of forced expiration involved earlier than those of inspiration. Pulmonary function tests typically disclose a decreased vital capacity, inspiratory reserve volume, and maximum breathing capacity. Because the disease process is slow in most cases, compensatory changes occur and a normal resting minute volume is generally maintained for long periods of time. Ventilation perfusion mismatch does not occur in limb-girdle muscular dystrophy, probably because of the uniformly diffuse disease process. Respiratory components affected include diaphragmatic and intercostal muscles. Pneumonia and respiratory failure can eventually shorten the life span. Some patients with mild phenotypes, however, remain mobile and have a normal life span.

Pathological changes in the myocardium in limb-girdle muscular dystrophy are seen in some forms more than others (478) and are generally similar to, but less severe than, in Duchenne dystrophy. Clinically overt cardiac involvement, however, is rare in limb-girdle muscular dystrophy except in a few subtypes.

A study has found that the Performance of the Upper Limb module, a functional upper extremity score traditionally used for Duchenne patients, can be applied and used for patients with limb-girdle muscular dystrophy as well with adequate reliability (171).

Additionally, findings indicate that miR-206 could be a prognostic indicator of disease progression. A study found that miR-206 is associated with disease severity for those with LGMD D4, R1, and D2 (392). The expression of miR-206 was elevated in those with limb-girdle muscular dystrophy in comparison to controls as a whole but was significantly elevated in those with severe disease in the specific aforementioned subtypes. This was characterized by worsened functional impairment when the expression was 50- to 80-fold higher as well as worsened atrophy confirmed by MRI.

Biological basis

Etiology and pathogenesis

Classification of limb-girdle muscular dystrophies. A reclassification of limb-girdle muscular dystrophy was proposed by the European Neuromuscular Center LGMD workshop study group in 2017. This new classification follows the formula: “LGMD, inheritance (R or D), order of discovery (number), affected protein” (477). Because of the definition of limb-girdle muscular dystrophy that needs to have been described in at least two different families, some conditions are no longer considered limb-girdle muscular dystrophy (for gene mutations with only one family described in the literature) (Table 1). Limb-girdle muscular dystrophy type 1 is now LGMD D and limb-girdle muscular dystrophy type 2 is now LGMD R. This review will describe the limb-girdle muscular dystrophies following the new classification. The description of the conditions no longer considered in current limb-girdle muscular dystrophy will be maintained for clarification purposes.

LGMD D (previously autosomal dominant [Type 1] limb-girdle muscular dystrophies). Autosomal dominant limb-girdle muscular dystrophies are much less common than recessive forms, accounting for fewer than 10% of all cases. Many of these entities have other neuromuscular or nonmuscular abnormalities linked to the same genes and in some cases the same gene mutations (Table 2).

LGMD D1 DNAJB6-related (previously limb-girdle muscular dystrophy 1D) (7q) – DNAJ homolog, subfamily B, member 6 (DNAJB6). DNAJB6 belongs to the DNAJ/HSP40 family of proteins, and it regulates molecular chaperone activity as a cochaperone (370). DNAJB6 is ubiquitously expressed, including in skeletal muscle, though with highest expression in the brain. Its best characterized function is the participation with the heat shock protein HSP70 in preventing aggregation of misfolded or otherwise aggregation-prone proteins, including polyglutamine containing huntingtin, alpha-synuclein, TDP-43, and Abeta42, through a process involving preferential interaction with misfolding forms of these proteins. One patient (with a known Phe93Leu DNAJB6 mutation) has been reported with pathologically confirmed frontotemporal dementia and no mutation in any of the known frontotemporal dementia genes. Whether this is a coincidence or other patients with DNAJB6-myopathy will also develop frontotemporal dementia with aging remains to be seen (554).

Mutations affecting the glycine/phenylalanine-rich (G/F) domain of DNAJB6 are most commonly reported, but mutations in J domain can also cause myopathy (441). These mutations likely interfere with the interaction between DNAJB6 and HSP70, resulting in defective antiaggregate properties and altered protein degradation system in cells – in other words, the mutations contribute to aberrant chaperone function (380; 442). There may be a genotype-phenotype correlation, with mutations affecting the amino acid in the more C-terminal part of the G/F domain (Pro96 and Phe100 in this) as well as mutations causing skipping of the entire G/F domain, correlating with a more distal phenotype compared to the more N-terminal amino acids (Phe89, Phe91, and Phe93) (429).

The p.A50V mutation in J domain has been associated with a distal calf-predominant phenotype whereas p.E54A mutation leads to a more proximo-distal phenotype (380). Onset can be very early (age 6) or late (6th decade). A retrospective study of 122 patients showed a mean age of onset of 29.7 years (156). Genotype appears to impact age of onset, weakness pattern, and the median time to loss of ambulation of around 34 years. Slowly progressive leg weakness is characteristic. A posterior-dominant pattern of lower limb muscle impairment with gluteus and truncal muscle involvement is typical (271). Distal predominant weakness is another manifestation of DNAJB6 mutations. Most patients with LGMD demonstrate no cardiac involvement. Dysphagia can be present (252). A family with progressive weakness starting in distal lower limb muscles and progressing to all other muscles including bulbar muscles (sparing extraocular muscles) has been described (428). Some patients are minimally symptomatic even in the seventh decade of life (567).

EMG shows myopathic changes and some patients can display pseudomyotonic or myotonic discharges (252). Muscle pathology demonstrates cytoplasmic inclusions and rimmed vacuoles. In muscle MRI, the radiological features of patients with DNAJB6 Phe93Leu mutations include soleus, adductor magnus, semimembranosus, and biceps femoris muscles in the early stages followed by medial gastrocnemius and adductor longus and later by vasti muscles of the quadriceps. Rectus femoris, lateral gastrocnemius, sartorius, gracilis, and the anterolateral group of the lower leg muscles are spared until late senescence (428).

In a preclinical mouse model of LGMD D1 DNAJB6 knockout mice, lithium chloride improved muscle size and strength. This could be a potential therapeutic option in the future (157).

LGMD D2 TNPO3-related (previously limb-girdle muscular dystrophy 1F) (7q32) –transportin 3 (TNPO3). Transportin 3 is a nuclear import receptor. The mutation discovered in the original Italo-Spanish family was a nonstop mutation (c.2771delA). Later, a missense mutation (c.G2453A) was found in a sporadic case and a frameshift mutation (c.2767delC) was found in a Hungarian family (503; 07). The mechanism of how these mutations in TNPO3 lead to disease is unknown but it may be related to its role in transporting serine/arginine-rich proteins that control precursor-mRNA splicing (181).

Penetrance of the mutation is incomplete and increases with age (148). In this condition, both shoulder and pelvic muscles are involved, but pelvic weakness predominates, and creatine kinase is mildly increased. In the upper extremities, the deltoid and triceps muscles are more affected, whereas in the lower extremities the quadriceps and anterior leg compartment are the most involved. Phenotypes are variable within the family; some patients have adult onset in the 40s, whereas others have a juvenile form starting before age 15 years or resembling a congenital myopathy. No dysarthria, cardiac involvement, or muscle contractures are seen. There is evidence of anticipation with earlier onset in children of affected parents. Specific clinical pointers and indicators for LGMD D2 are skeletal abnormalities, such as arachnodactyly, pes cavus, and mild Achilles tendon retraction. Macroglossia, mild facial weakness, calf hypertrophy, gynecomastia, dysarthria, partial ophthalmoparesis, neck extension, and flexor weakness (181) were only occasionally observed. Dysphagia was found in 8 of 29 patients in a case series. A rapid worsening of symptoms was reported to have occurred following intense physical exercise, alcohol intake, long periods of inactivity, benzodiazepine overuse, and pregnancy in some cases.

Muscle MRI shows the atrophy is more prominent in lower than in upper extremities, preferentially in the vastus lateralis and posterior leg compartment (398).

There can be sparing of the gracilis and rectus femoris muscles (181). Muscle biopsy findings include increased fiber size variability, fiber atrophy, and acid-phosphatase-positive vacuoles (79), ragged red fibers, cytoplasmic bodies, and enlarged mitochondria with procrystalline inclusions (181). Rod-like structures and other changes suggest a derangement of the cytoskeletal network.

LGMD D3 HNRNPDL-related (previously limb-girdle muscular dystrophy 1G) (4p21) – HNRPDL. HNRNPDL is a heterogeneous ribonucleoprotein (hnRNP) family member that has been shown to function in mRNA biogenesis and mRNA metabolism, including alternative splicing, mRNA nuclear export, translational regulation, and turnover. HNRNPDs participate in the splicing of specific exons in pre-mRNA of transcripts from important muscle-related genes (524). The disease has been found in four families so far with mutations affecting the same codon (c. G1132C/A, p.D378H/N) in HNRNPDL (470; 524; 38; 480). The mutation may lead to a loss of function, impacting hnRNPDL self-assembly properties, and accelerating protein aggregation in muscle cells (26).

The phenotype includes pure limb-girdle presentation starting in lower extremity, distal lower limb weakness, and oligosymptomatic cases. Limitation of finger and toe flexion is also a characteristic of this condition, although not always present (470; 480). Weakness usually starts in the second to fifth decade. Early onset (younger than 50-years-old), cataracts, and diabetes can also be present. Myotonia is absent. In a Spanish family with a scapulo-peroneal phenotype, cognitive impairment was another characteristic (522).

Scapular winging was characteristic in a case series (38).

In Italy, a woman with a previously reported pathogenic variant c.1132G > C p.(Asp378Asn) in the HNRNPDL gene experienced diaphragmatic weakness requiring noninvasive ventilation during hours of sleep, a unique case because respiratory muscles are not usually involved in LGMD D3 (316).

Muscle MRI shows involvement of vastus muscles, with partial sparing of rectus femoris and biceps femoris and complete sparing of adductor longus (480).

Muscle biopsy shows a predominantly myopathic histopathological pattern associated with rimmed vacuoles and autophagic vacuoles (38).

LGMD D4 calpain3-related (previously limb-girdle muscular dystrophy 1I) (15q) – Calpain-3 (CAPN3). Calpain 3 mutations are one of the most common causes of limb-girdle muscular dystrophy (LGMD R1 calpain3- related or LGMD 2A in the prior classification).

Interestingly, although CAPN3 mutations usually cause autosomal recessive limb-girdle muscular dystrophy (LGMD R1), there have been reports of patients heterozygous for the c.643_663del21 mutation who presented with autosomal dominant limb-girdle muscular dystrophy (LGMD D4). These patients reported variable weakness in their 40s or 50s, with myalgia, back pain or hyperlordosis. Pelvic girdle muscles were affected with adductor and hamstring sparing (different from LGMD R1). Creatine kinase was normal to elevated, independent of weakness severity. Electromyography and muscle biopsy were normal to mildly myopathic. Western blot muscle CAPN3 expression was reduced (528; 323; 360).

Further missense mutations in CAPN3 have been characterized for LGMD D4, including c.700G> A, [p.(Gly234Arg)], c.1327T> C [p.(Ser443Pro], c.1333G> A [p.(Gly445Arg)], c.1661A> C [p.(Tyr554Ser)], and c.1706T> C [p.(Phe569Ser)] (186). Another missense variant is c.1715G>C p.(Arg572Pro) and another deletion mutation of CAPN3 discovered is c.643_663del (529).

A study using next-generation sequencing in 4656 patients with limb-girdle muscular dystrophy phenotype found an in-frame deletion c.598_612del15 in the CAPN3 gene without a second pathogenic variant in 16 patients, indicating another potential autosomal-dominant form of calpainopathy in these cases (360). For more details on CAPN3 function and clinical features, see LGMD R1 description.

LGMD D5 collagen 6-related (previously Bethlem myopathy dominant) (21q, 2q) – collagen type VI subunits alpha-1, 2, and 3 (COL6A1, COL6A2, COL6A3). This condition is caused by mutations to three genes encoding different subunits of collagen type VI. Two of them are on chromosome 21q (alpha-1, alpha-2) and one on chromosome 2q (alpha-3) (248).

In 1976, Bethlem and Van Wijngaarden described an autosomal dominant, early-onset but relatively benign limb-girdle muscular dystrophy associated with flexion contractures in many members of three unrelated Dutch families. The progression was extremely slow, with periods of arrest for several decades; the ability to walk was preserved until old age. Ankle and elbow joints seemed preferentially affected and long finger flexor contractures were usually the first signs. Serum creatine kinase was normal or slightly elevated, EMG showed a myopathic pattern, and muscle biopsy disclosed nonspecific myopathic alterations.

Onset is earlier than in typical limb-girdle muscular dystrophy; nearly all patients demonstrate weakness or contractures before 2 years of age, but progression is slow and continues into adulthood (247). More than two thirds of patients over 50 years of age are wheelchair bound (247; 264). Three family members heterozygous for c.2107A>C p.T703P mutation in the COL6A2 gene had symptom onset at 40 to 60 years of age and the diagnosis was suspected only based on characteristic muscle MRI findings (251).

Similar to other dominant limb-girdle muscular dystrophy syndromes, an allelic recessive disorder was described: Ullrich scleroatonic muscular dystrophy. This is an autosomal recessive form of congenital muscular dystrophy with distinctive proximal joint contractures, distal joint hyperextensibility, scoliosis, and normal intelligence (221; 515). Later, autosomal dominant and recessive patterns have been observed in both Ullrich and Bethlem phenotypes. Intermediate manifestations are also common (264; 71; 561). A large clinical variability, including intrafamilial variability, has been seen (121; 561). The recessive form of collagen VI-related dystrophies is designated as LGMD R22.

Muscle MRI in COL6 in patients with Bethlem myopathy and Ullrich congenital muscular dystrophy characteristic findings that aid in the diagnosis. In patients with Bethlem muscular dystrophy, the vasti muscles show a rim of hyperintensity at the periphery of the vastus lateralis and hyperintensity at the center of the rectus femoris (central shadow). There is relative sparing of the sartorius, gracillis and adductor longus muscles. In the calf, there is a rim of hyperintensity between the soleus and the gastrocnemius. The MRI findings are similar in Ullrich congenital muscular dystrophy, except for the presence of a more diffuse muscle involvement (331; 138).

The use of skin biopsy with COL6 staining is postulated to facilitate diagnosis (82). However, COL6 expression can be only mildly reduced in Bethlem myopathy and variable in Ulrich muscular dystrophy, making diagnosis difficult. Some skin physical exam findings, such as follicular hyperkeratosis and keloid formation, may provide cues to possible COL6 deficiency in the right clinical context (280).

Interestingly, mutations in COL6A3 have also been associated with a form of isolated recessive dystonia (DYT27) (301).

LGMD R (previously autosomal recessive [type 2] limb-girdle muscular dystrophies). This category includes a group of autosomal recessive disorders, although some may appear to be sporadic. Proximal muscles are more severely affected; cardiac and bulbar muscles are typically but not exclusively spared. These disorders include the majority of limb-girdle muscular dystrophy cases. There is considerable clinical overlap between entities and diagnosis on purely clinical grounds remains problematic, although some differing characteristics will be highlighted where appropriate.

LGMD R1 calpain3-related (previously limb-girdle muscular dystrophy 2A) (15q) – Calpain-3. LGMD R1 was the first muscular dystrophy shown to be caused by an enzymatic abnormality rather than a structural muscle protein defect. Calpain-3 protein likely functions as a homodimer and is a calcium-activated nonlysosomal cysteine protease that plays a role in muscle regeneration, sarcolemmal repair, sarcomere remodeling, cytoskeleton regulation, and calcium homeostasis (422; 126; 210). Calpains are found in all mammalians and many plant tissues, but the CAPN3 isoform is predominantly found in muscle.

CAPN3 can be detected in three compartments (myofibrillar, cytosolic and membrane fractions. The myofibrillar fraction is bound to titin, which stabilizes it. The cytosolic fraction is stabilized by the Platform Element for Inhibition of Autolytic Degradation (PLEIAD) protein. The membrane fraction exhibits more activity and is concentrated at the triad, where it colocalizes with a protein complex including RyR1, CaMK, and aldolase (137; 375) and works as a structural protein rather than a protease. Studies have shown that calpain-3 can cleave the C-terminus of filamin-C in vitro and that this modification may decrease the ability of filamin-C to bind to delta- and gamma-sarcoglycans (197). The secondary CAPN3 deficiency in titin gene defects (LGMD2J) suggests that the pathomechanisms of the diseases are linked. Studies in yeast have identified myospryn (linked to hereditary cardiomyopathy) as a binding partner for both M-band titin and CAPN3 (440). The protein may also play a role as a switch in muscle remodeling (108). Calpain-3 may also be related to dysferlin, the protein involved in LGMD R2, by a common protein AHNAK, which is involved in subsarcolemmal cytoarchitecture and membrane repair. Calpain-3 cleaves AHNAK, which in the fragmented form loses its affinity for dysferlin (229).

Studies in calpain-3 knockout mice have shown that lack of calpain-3 leads to reduced ryanodine receptor (RyR1) expression, abnormal calcium 2+/calmodulin-dependent protein kinase II (Ca-CaMKII)-mediated signaling, attenuated calcium release, and impaired muscle adaptation to exercise (114). Calmodulin is a positive regulator of CAPN3 activity. Calpain-3 deficient myotubes show increased degradation of sarco/endoplasmic reticulum Ca2+ ATPases (SERCA) proteins (502). Studies in muscle tissue suggest that the dystrophic processes in LGMDR1 associate with oxidative stress, increased protein ubiquitinylation, and activation of NF-kB p65. In LGMD R1 muscle protein, ubiquitinylation may occur through the activation of MuRF 1 and MAFbx. NADPH oxidase appears to be one possible source of oxidative stress in these dystrophic muscles. Calpain-3 also appears to be important for muscle regeneration, comparing LGMD R1 patient samples from others with LGMD R9 and Becker muscular dystrophy (211). In LGMD R1, frizzled related protein (FRZB) is upregulated and there appears to be a reciprocal relationship between FRZB and CAPN3 (76).

First reported in 37 affected members of two large Amish kindreds (240), this form of muscular dystrophy was later reported in families from the French Reunion Island near Madagascar (31; 150) and in Brazilian families (389). A genetic epidemiology study in northeastern Italy found a prevalence of 9.47 per million, suggesting that LGMDR1 is one of the most common autosomal recessive disorders (144). Several different mutations in the CAPN3 (calpain-3) gene have been reported in affected members of these genetically unrelated families. The entity is a common cause of limb-girdle muscular dystrophy; close to 500 distinct mutations have now been identified, although a small number of mutations underlie the majority of cases (188; 141) (Leiden muscular dystrophy database). In some populations, mutations in this gene account for 28% to 50% of limb-girdle muscular dystrophy cases if sarcoglycanopathies and dystrophinopathies are excluded (91; 147). Some particularly common mutations include the c.2120A>G mutation in the Chinese population and the c.550del in European populations (565). LGMD R1 is the most common form in France, with 10 to 70 cases per million people (315).

Age at onset ranges from 2 to 45 years; loss of ambulation occurs about 10 to 30 years after onset of weakness, from early adulthood to the seventh decade (usually between 10 and 45 years of age). There is no correlation between age of onset and time of wheelchair confinement (431). The type of mutation was not entirely predictive of disability, though patients with two null mutations seem to be less ambulatory than patients with one missense mutation. The weakness usually starts in the pelvic girdle with later involvement of the shoulder girdle, or less commonly starts in the shoulder girdle (usually milder form). The characteristic pattern is muscular atrophy of pelvic girdle sparing hip abductors even in late stages, scapular winging, abdominal laxity, frequent contractures, and preserved respiratory function (403; 65). Early involvement of hip adductors and hamstring is common. Facial and neck muscles are usually spared. Calf muscle hypertrophy and cardiac involvement are uncommon. Creatine kinase is often elevated but not to the levels seen in the sarcoglycanopathies, dysferlinopathies or dystrophinopathies. In patients with long disease duration, nonambulatory, and with normal CK levels, the frequency of respiratory dysfunction increases (up to 20%) (348). Respiratory failure usually occurs in advanced disease, although one case with marked respiratory muscle weakness in a still-ambulatory patient has been described (322). There is usually no cardiac involvement.

Pizzanelli and colleagues reported a young girl with LGMDR1 and coexistent generalized epilepsy (401). Tsao and Mendell describe a boy with absence seizures, a normal brain MRI, and partial calpain deficiency (505). There is one report of late-onset foot drop as a presenting sign (63). Because of this heterogeneity, it is currently impossible to definitively distinguish this form of dystrophy from other forms of limb-girdle muscular dystrophy on clinical features alone. Late-onset myopathy and camptocormia were described in a calpainopathy carrier (291).

There is considerable variation in disease severity, including some with isolated hyperCKemia and others with predominantly distal myopathy (147). An early and transient feature in LGMDR1 may be eosinophilic myositis, which has been documented only in young patients with increased creatine kinase (CK) levels and peripheral blood hypereosinophilia. Some patients with LGMDR1 may present with a pseudometabolic myopathy, with asthenia, myalgia, exercise intolerance, proximal muscle weakness, and excessive lactate production. Asymptomatic hyperCKemia (5 to 80 times normal values), usually observed in children or young patients, is the preclinical stage of the disease and may persist for decades. CK levels gradually decrease with the progression of muscle atrophy and weakness (140). The disease appears clinically more heterogeneous among the Amish families than among the Reunion Island families, but cases have been seen in numerous countries worldwide (403; 144; 17; 216; 366; 417; 540; 02; 397; 418; 496; 548; 230). Interestingly, the disease seems to have slower progression in the Indian population in comparison to Europeans and missense mutations seem to confer an overall better prognosis in comparison to nonsense mutations (390). Muscle MRI shows selective involvement of thigh muscles in the lower limbs, mainly of the adductors, hamstrings, and gluteus minimus. Sartorius, gracilis, quadriceps, biceps short head, and iliopsoas are relatively spared. At calf level, the involvement is predominant at the soleus muscle and of the medial head of the gastrocnemius with relative sparing of the lateral head of gastrocnemius and other muscles belonging to the anterolateral compartment of the leg. As muscle MRI may show a typical pattern of involvement even in the “contracted” phenotype of LGMD2A and may help to differentiate it from other clinically similar muscular dystrophies such as Emery-Dreifuss and Bethlem myopathy, imaging might help to tailor further genetic testing in these phenotypically similar patients (330). Like collagen VI-related disorders, a high signal observed at the central area of muscles on MRI is associated with longer and more severe disease course (21).

Muscle biopsy findings in patients with LGMDR1 can vary from the usual dystrophic pattern, with prominent lobulated fibers and scanty inflammatory infiltrates, to one with eosinophilic myositis or macrophage-rich regional inflammatory infiltrates, which can be confused for an autoimmune disease but does not respond to immunosuppressive treatment (453). Mitochondrial abnormalities with COX-negative muscles fibers, ragged red fibers and mitochondrial depletion have also been described (130).

At present, protein immunostaining of muscle biopsy samples is not highly reliable, and Western blotting or genetic testing is needed to confirm the diagnosis. In a review of 208 cases based on protein and genetic analysis, it was found that the probability of a gene defect is high with complete protein deficiency (84%) and declines with increasing protein expression (141). Whereas some calpain-3 mutations alter the level of protein expression, others alter the autocatalytic enzyme activity without affecting protein expression (145). Fanin and colleagues screened 148 muscle biopsy specimens with normal calpain-3 levels and found that 11% of samples had abnormal autolytic function (143). Some patients with limb-girdle muscular dystrophy-R1 will be missed if they are tested by immunohistochemistry or Western blotting only. Fanin and colleagues conducted a quantitative analysis of calpain-3 protein in 519 muscles from patients with unclassified limb-girdle muscular dystrophy, unclassified myopathy, and hyperCKemia as well as a functional assay of calpain-3 autolytic activity in 108 cases with limb-girdle muscular dystrophy. Coincident genetic testing found 94 LGMD2A patients, carrying 66 different mutations (6 newly identified). The probability of diagnosing calpainopathy was very high in patients showing either a quantitative (80%) or a functional calpain-3 protein defect (88%). They concluded that reduced or absent calpain-3 or lost autolytic activity had a high predictive value for a genetic diagnosis. These methods are proposed to be desirable because of the laborious task of identifying one of the multitudes of genetic defects in this condition (146). Only 70% of patients with recessive calpainopathy have quantitative calpain-3 deficiency by immunoblot. The remaining 30% of patients may have mutations that alter protein function but not quantity, manifesting with compromised autolytic calpain-3 activity (148). In a small proportion of cases, calpain-3 protein deficiency can be secondary to a primary defect of other proteins, such as dysferlin and titin, or to its artificial degradation as a consequence of inadequate tissue treatment. Many other diverse disorders have been associated with, likely secondary, calpain-related dysfunction, but are not due to gene mutations. One intriguing example is a suggested secondary deficiency of calpain-3 in patients with myoglobinuria from eating toxic quail meat (359).

Other gene defects may secondarily affect calpain-3 function and expression as well. The abnormally expressed genetic region mutated in facioscapulohumeral muscular dystrophy (FRG1) affects other downstream genes including Rbfox1 that appears to secondarily affect calpain-3 expression. Facioscapulohumeral muscular dystrophy (FSHD) and LGMD2A share some phenotypic features. In FRG1 mice and FSHD patients, the calpain-3 isoform lacking exon 6 is increased. Also, Rbfox1 knockdown and over-expression of the calpain-3 E6- isoform inhibit muscle differentiation. The results suggest aberrant expression of an altered calpain-3 protein through dysregulated splicing (400).

Interestingly, although CAPN3 mutations usually cause autosomal recessive LGMD R1, there have been reports of patients heterozygous for the c.643_663del21 mutation who presented with autosomal dominant LGMD (see LGMD D4), which tends to be less severe than the recessive form.

LGMD R2 dysferlin-related (previously limb-girdle muscular dystrophy 2B) (2p) – dysferlin. This subtype of limb-girdle muscular dystrophy is associated with defects in the DYSF gene that encodes the protein dysferlin and is expressed in adult mammalian cardiac and skeletal muscles as well as multiple nonmuscle tissues, including kidney, brain, and lung. Bashir and colleagues reported the initial linkage of this limb-girdle muscular dystrophy to 2p in Palestinian and Sicilian families (25), but patients have been identified in numerous countries. Dysferlin appears to play an important role in sarcolemmal repair, especially sarcolemmal resealing. In normal skeletal muscle cells, injury leads to a process of Ca2+-dependent membrane repair that involves membrane patches enriched in dysferlin (20). In dysferlin-null mice, this repair process is defective, and the mice develop progressive muscular dystrophy (20). A mouse model also displays early, severe, and progressive diaphragm involvement—a finding not appreciated in humans (24). Subcellular labeling of dysferlin also colocalizes with the developing T-tubule system in mice; dysferlin is necessary for correct T-tubule formation; and dysferlin-deficient skeletal muscle is characterized by abnormally configured T-tubules (269; 224). Most of the dysferlin present in mature skeletal muscle fibers concentrates in the T-tubules, where it stabilizes Ca2+ release at the triad junction when muscle is injured either in vitro or in vivo (305). Dysferlin is also thought to have a role in myogenesis, angiogenesis, microtubule dynamics, cytokine secretion, lysosome exocytosis, acid sphingomyelinase secretion, and phagocytosis (95). In mice, dysferlin deficiency confers increased susceptibility to coxsackievirus-induced cardiomyopathy (538). Dysferlin also may play a role in cholinergic signaling in the neuromuscular junction (273). There is also activation of the ubiquitin proteasome system (UPS) and autophagy programs in dysferlinopathy partly due to regeneration and an inflammatory reaction (142). Humans also produce an alternate splice variant lacking one exon (delta17), which is replaced by the full-length version during muscle development but is the predominant form in normal peripheral nerve (433).

Dysferlin is normally present in muscle membrane and is lost in limb-girdle muscular dystrophy R2. Miyoshi myopathy, a rare disorder but the most common form of distal-onset myopathy, is an allelic disorder with genetic defects not only in the same DYSF gene but also often with identical mutations (339). The defect is a common cause of distal myopathy either with severe posterior compartment (gastrocnemius) involvement in Miyoshi myopathy or with distal myopathy with anterior tibialis onset (DMAT) (433). A series of Spanish patients were found to have identical dysferlin mutations and three different phenotypes: limb-girdle muscular dystrophy, Miyoshi myopathy, and anterior compartment atrophy (525). Other clinical presentations in patients with dysferlin mutations include proximodistal weakness (more severe), exercise intolerance, and asymptomatic hyperCKemia.

LGMDR2 prevalence is 1/100 000 to 1/200 000 people. Onset is often later than in other recessive limb-girdle muscular dystrophy forms, usually after age 15 (as late as 7th decade) with early lower extremity involvement and difficulty in toe walking and running. There is preservation of hand and neck muscles at the late stage and lack of facial muscle weakness or dysphagia. There is rare cardiac dysfunction. Respiratory function declines with disease duration. Loss of ambulation occurs at a mean of 20-year disease duration (489). Joint contractures commonly affecting ankles, knees, and elbows can be present in 36% of patients. Distal lower limb muscle atrophy is seen in 71% of cases. Muscle pseudohypertrophy can be seen in 11% of cases, usually in distal lower extremity muscles. Up to 19% of patients report an above-average sporting ability before the onset of symptoms (207).

A wide variety of phenotypes have been observed in the series of DYSF mutations characterized to date (68). Ueyama and colleagues found that calf involvement with sparing of the tibialis anterior is common to both diseases but is the dominant feature in Miyoshi myopathy, whereas proximal muscle weakness dominates in limb-girdle muscular dystrophy (507). Late-onset muscular dystrophy linked to dysferlin mutations has also been reported in a 73-year-old man with proximal muscle weakness (268) and another elderly patient with isolated calf weakness and atrophy and markedly elevated creatine kinase (255). The phenotype does not appear to affect progression or prognosis (387). Interestingly, on MRI studies the adductor magnus and gastrocnemius medialis were the first to be impaired in both phenotypes. Several other phenotypes have been described.

Rarely, a rigid spine phenotype can develop. A case report from Japan describes a patient with LGMDR2 and chorea (488). An LGMDR2 patient was reported with coexisting sarcoidosis and autoimmune Addison disease, suggesting a possible link between LGMDR2 and autoimmunity (456). Another report describes a patient with LGMDR2 and minimal change nephropathy with absent dysferlin in the renal glomeruli (239). Rosales and colleagues noted some characteristic features in a cohort of 21 patients (427). A distinct "bulge" of the deltoid muscle in combination with other typical findings was noted. Respiratory weakness and cardiac involvement are atypical. However, some LGMDR2 patients progressively develop dilated cardiomyopathy with reduced left ventricular ejection fraction and interstitial fibrosis. Some patients without cardiac symptoms have mild left ventricular hypertrophy. A case misdiagnosed and treated for refractory polymyositis was reported (526).

Different phenotypes can occur within families, but affected siblings typically have the same disorder (465; 462). Fanin and colleagues found that 60% of biopsied patients with distal myopathy had a dysferlin mutation, but only 1% of patients with undiagnosed limb-girdle muscular dystrophy were positive (147). However, defects in the DYSF gene are the second most common cause of limb-girdle muscular dystrophy in some populations. Because of the prevalence of dysferlinopathies, Fanin suggests screening for dysferlin mutation-suspected carriers and for asymptomatic patients with idiopathic hyperCKemia (143).

A muscle MRI study showed that head and cervical muscles, levator scapula, trapezius, pectoralis minor, popliteus, piriformis, posterior forearm compartment, and transversus abdominis muscles are usually spared. On the other hand, subscapularis, lumbaris erector spinae, gluteus minimus, tensor fasciae latae, obturator externus, quadriceps, semimembranosus, semitendinosus, biceps femori, hip adductors, and all leg muscles (except popliteus) are usually involved (184). A case report demonstrated some unusual fatty degeneration in the lumbar paraspinalis in an LGMD R2 patient, detected on MRI (262).

Muscle biopsies may show inflammatory response more often than in other forms of limb-girdle muscular dystrophy and serum creatine kinase is typically markedly increased. These features can lead to confusion with an inflammatory myopathy. Inflammasome upregulation was demonstrated in dysferlin-deficient muscles, suggesting activation of proinflammatory cytokines, including IL-1beta and IL-18.

In addition, clinically affected heterozygotes have been reported in two Spanish mutations (234). Both patients had clinical weakness, elevated CK, and abnormal muscle biopsy and muscle MRI but normal dysferlin PCR and no coincident unrelated mutation; both had homozygous affected relatives. A different family with pauci-symptomatic (cramps, mildly elevated CK) heterozygous carriers has also been described (243).

Genetic testing should replace the need for muscle biopsy. There are currently over 250 different gene defects reported (Leiden muscular dystrophy database). In fact, new variants of DYSF gene mutations in LGMD R2 continue to be reported, including a novel heterozygous variant that involves a frameshift mutation of c.4010delT (288). Absence of the protein is diagnostic for LGMDR2 (09). However, there are patients with reduced but not absent protein, or dysfunctional dysferlin (498) and some with increased dysferlin expression in Western Blot due to a pathological retention of mutated polypeptide in the cytoplasms. For this reason, immunohistochemistry in biopsy samples can be unreliable, and DNA sequence testing is recommended. Dysferlin western blot analysis in monocytes has been proven to be highly predictive of primary dysferlinopathy provided that protein quantity is lower than 10%; the amount of dysferlin was evaluated to be less than 11% in affected patients, 24% to 78% in heterozygous carriers, and greater than 75% in healthy individuals. However, there are false-negative results (139). A nonquantitative immunoassay that may be used at centers with limited resources for detection of dysferlinopathies from peripheral blood samples has been developed (100).

Dysferlin expression in muscle can also be reduced in patients with sarcoglycan, dystrophin, caveolin, or calpain mutations. Next-generation sequencing in 90 patients with dysferlin deficiency in muscle revealed that 70% had DYSF mutations, 10% had CAPN3 mutations, 2% had CAV3 mutations, 3% had mutations in other genes (in single patients), and 16% did not have any identified mutations (238).

Many animal models are in use to search for potential treatment. In a murine model of LGMB2B, human adipose-derived stromal cells (HASCs) were inserted without immunosuppression of the animals. The dystrophic mouse muscle was able to fuse with HASCs, express human muscle proteins, and improve motor function in the animals (523). Wang and colleagues developed an in vitro assay based on the finding that membrane blebbing in a dysferlin-deficient mouse and human myocytes model in response to hypotonic shock requires dysferlin (537). Using this model and the nonsense suppression drug, ataluren (PTC124), the group was able to induce read-through of the premature stop codon in a patient with a dysferlin R1905X mutation and produce sufficient functional dysferlin (approximately 15% of normal levels) to rescue myotube membrane blebbing. Also, it has been shown that antisense oligonucleotide-triggered exon skipping is possible in myoblasts generated from control and patient MyoD transduced fibroblasts; high efficiency skipping of exon 32 was demonstrated in this model (543). This strategy may prove to be useful in other exons (284). Another approach is using peptides that might restore aberrant dysferlin function. A German group has created short peptides derived from the dysferlin protein that seem to reduce folding and aggregation errors in the endoplasmic reticulum of myoblast cells derived from patients following laser induced membrane injuries (450). In a dysferlinopathy mouse model, treatment with recombinant human MG53 protein (rhMG53) enhanced muscle membrane integrity independently of the canonical dysferlin-mediated, Ca2+-dependent pathway known to be important for sarcolemmal membrane repair, suggesting a possible therapeutic option (195). AMP-activated protein kinase (AMPK) complex was found to be a potential therapeutic target for dysferlinopathy. AMPK complex interacts with dysferlin and AMPK activator metformin was shown to improve the muscle phenotype in zebrafish and mouse models of dysferlin deficiency (374).

Conventional pharmacological treatment attempts are less encouraging. A blinded, placebo-controlled trial of deflazacort failed to show benefit in a cohort of 25 dysferlinopathy patients randomly treated for 6 months following 1 year of quantified natural decline without treatment, and there was a trend of worsening muscle strength under deflazacort treatment, which recovered after its discontinuation (535). The use of dissociative steroid compounds such as vamorolone (that retains therapeutic corticosteroid effect while reducing side effects) stabilized dysferlin- deficient muscle cell membrane and improved repair of dysferlin-deficient mouse (B6A/J) injured myofibers, indicating its potential use in future clinical trials (468). Ibuprofen was detrimental to muscle function in dysferlin-deficient mice (98).

Dysferlin-null mice exhibited a cholinergic deficit manifested by a progressive, frequency-dependent decrement in their compound muscle action potentials (CMAP) following repetitive nerve stimulation, which was reversed by pyridostigmine, suggesting a possible treatment avenue for this condition (273). Another potential treatment was diltiazem, which reduced eccentric contraction-induced t-tubule damage, inflammation, and necrosis and resulted in a concomitant increase in postinjury functional recovery in dysferlin-deficient mice (261). However, an improved study protocol did not find diltiazem to have any effect on contraction-induced muscle damage in dysferlin-deficient mice (32).

Most encouraging is work to insert a functional copy of the dysferlin gene through viral vector. The full gene is too large to insert, but a cDNA construct was transfected into a mouse model through an adeno-associated virus (AAV) gene transfer and successfully restored function (192). The approach may be suitable for human trials.

Human serum exosomes also carry dysferlin protein and improved membrane repair in dysferlin deficient myotubes. This could be a strategy to deliver dysferlin to affected muscles (120).

Another approach has been the injection of plasmids encoding dysferlin to hindlimbs of dysferlin-knockout mice (308; 193); this could potentially be applied to humans in the future.

LGMD R3, 4, 5, and 6 (previously limb-girdle muscular dystrophy 2C-F – sarcoglycanopathies). Sarcoglycans are glycoproteins needed for the stability of the muscle membrane. The four primary protein forms (alpha, beta, gamma, and delta) form a complex that provides support and signaling functions for the muscle membrane. The complex supports the association between dystrophin and beta dystroglycan (377). All four of these dystrophin-associated glycoproteins (DAGs) have been implicated in specific forms of limb-girdle muscular dystrophy. A distinct epsilon subunit replaces alpha-sarcoglycan in smooth muscle and in some striated muscle cells (476). Zeta-sarcoglycan, identified in mouse smooth and striated muscle, is another subunit of the sarcoglycan complex; levels were reduced in murine muscular dystrophy (544). This protein is also found in brain tissue. Epsilon sarcoglycan mutations lead to a form of myoclonus-dystonia syndrome. Mutations in any of the four main subunits often disrupt the sarcoglycan complex as a whole, leading to decreased biochemical function, typically loss of staining on biopsy samples, and impairment of cytoskeletal-sarcolemmal integrity (30). In fact, the disease in affected individuals is related to the level of residual function of the glycoprotein complex (06). Complete loss of the tetramer will result in earlier onset and more severe phenotype. Complete loss of sarcoglycan manifest from 3 to 15 years of age and patients are typically wheelchair-bound by age 15. Initially, this was thought to be due to underexpression of the sarcoglycan-sarcospan complex because of the mutant sarcoglycan subunits and marked by decreased immunohistochemical staining. McNally and colleagues subsequently demonstrated that loss of the gamma subunit in a knockout mouse model allowed partial retention of the alpha and beta subgroups (199). Studies by Crosbie and colleagues have shown that even when a sarcoglycan-sarcospan complex forms in the setting of a defective gamma subunit, the mutant form is not sufficient to prevent disease. This suggests an even more complex protein-protein interaction than initially thought (101). Beta-sarcoglycan appears to play an initiating role in complex formation and its association with delta-sarcoglycan is essential for proper localization of the complex to the cell membrane. Alpha-sarcoglycan is incorporated at the final stage by interaction with gamma-sarcoglycan (460).

Partial loss of sarcoglycan delays onset until the teens or early adulthood. Interestingly, the primary deficiency of dystrophin in Duchenne dystrophy can also result in secondary deficiency of dystrophin-associated glycoprotein components (369) and dystrophin must be shown to be normal before a diagnosis of sarcoglycanopathy can be made on immunohistochemical grounds. Overall mutations in the four sarcoglycans represent roughly 10% of teen- or adult-onset cases (91) with type alpha sarcoglycanopathy occurring most commonly.

Mutations of beta or delta sarcoglycan often have associated cardiomyopathy. This cardiomyopathy appears to be initiated by perturbation of the sarcoglycan complex in vascular smooth muscle cells that causes vascular constrictions and ischemic events. Notably, both the constrictions in the cardiac vasculature and the cardiomyopathy can be blunted experimentally with the calcium-channel blocker verapamil (97). In contrast, the skeletal muscle abnormalities seen in mice lacking beta or delta sarcoglycan can be rescued by adenovirus-mediated gene transfer into skeletal muscle but not vascular smooth muscle, so it appears that abnormalities of the sarcoglycan complex within skeletal muscle itself are sufficient for the resultant skeletal muscular dystrophy (127).

Muscle MRI shows similar findings in all sarcoglycanopaties: quadriceps gradient with more severe proximal involvement, adductor longus medial sparing, relative sparing of tibialis posterior, flexor digitorum longus and tensor fasciae latae, and hypertrophy of either sartorius or gracilis (492).

LGMD R3 alpha-sarcoglycan-related (previously limb-girdle muscular dystrophy 2D) (17q) – alpha-sarcoglycan (formerly known as adhalin). McNally and colleagues described a French kindred with autosomal recessive limb-girdle muscular dystrophy in which the 50-kd dystrophin-associated glycoprotein adhalin was absent in muscle fibers (326). The adhalin (alpha-sarcoglycan-SGCA) gene was initially mapped to chromosome 17q21 region and later cloned and identified. Adhalin (alpha-sarcoglycan) is an integral component of the dystrophin-associated glycoprotein complex (557). Although mostly expressed in skeletal muscle, adhalin alpha-sarcoglycan mRNA is also present in heart and lung, although Adhalin alpha-sarcoglycan in lung may reflect contamination by smooth muscle. This is the most common of the sarcoglycanopathies, with one mutation accounting for roughly half of all known cases (124).

The clinical severity of myopathy in patients with alpha-sarcoglycan mutations varies strikingly. The most severe phenotypes occur in patients homozygous for null mutations, who express no alpha-sarcoglycan in muscle fibers. Hackman and colleagues identified 11 Finnish patients with LGMD R3; 10 were homozygous for the R77C mutation and one patient was a compound heterozygote with one copy of the R77C allele (200). Patients presented between ages 5 to12 years and all were able to run as young children. They may resemble the X-linked Becker or Duchenne phenotype of dystrophinopathy, including calf hypertrophy, distribution of muscle weakness, and elevated CK; however, cardiac involvement is absent in alpha-sarcoglycanopathy. Patients from a German kindred heterozygous for a 371 T --> C mutation had clinical manifestations ranging from a severe Duchenne-like phenotype in the homozygous index case to mild or moderate weakness including scapular winging in 7 of 12 heterozygote carriers (160). Missense mutations cause relatively milder phenotypes and variable residual adhalin (alpha-sarcoglycan) expression (399). Severity may be as mild as asymptomatic hyperCKemia and patients may also present with very late-onset cases (373). A case presenting with myoglobinuria has also been described (80). Immunohistochemical staining showing alpha-sarcoglycan deficiency can confirm the diagnosis as well as mutation analysis of the SGCA gene. In countries with a high incidence of LGMD R3, SGCA gene analysis should be strongly considered in some boys and certainly in girls with a Duchenne phenotype. In some cases, immunohistochemical staining is normal but Western blot shows decreased alpha sarcoglycan levels (70).

Muscle MRI shows severe involvement of proximal lower extremity muscles and relative sparing of leg muscles until late stages (70). Paraspinal, pelvic, and glutei muscles can be preferentially affected in mild cases (187).

Gene therapy is being explored in murine models, and several promising studies have been published. Using adeno-associated virus type, Rodino-Klapac and associates injected human alpha-sarcoglycan gene into the tibialis anterior of mice using several different promoters (425). They found that sustained, nontoxic levels of alpha sarcoglycan were produced under muscle creatine kinase promoters. Mendell and colleagues conducted a double-blind, randomized controlled trial of adenovirus-associated virus gene transfer into the extensor digitorum brevis muscles of a cohort of affected patients (328). The full sarcoglycan complex was restored in all subjects, and muscle fiber size was increased in one subject at 3 months. Antibodies against the virus developed, but no signs of apoptosis were evident. Mitochondrial biogenesis was impaired in patients and mice with LGMD R3 and was associated with impaired OxPhos capacity. The histone deacetylase inhibitor trichostatin A restored mitochondrial biogenesis and enhanced muscle oxidative capacity (381).

Another possible treatment strategy is employing stem cells that may express alpha-sarcoglycan. Tedesco and colleagues have reprogrammed fibroblasts and myoblasts from patients with LGMD R3 to generate human-induced pluripotent stem cells (iPSCs) and virally transfected the cells to produce alpha-sarcoglycan. Cells transplanted into alpha-sarcoglycan-deficient mice demonstrated restoration of protein expression and reversal of the phenotype (495).

In myotubes from a patient with LGMD R3, treatment with cystic fibrosis transmembrane regulator correctors induced the proper relocalization of the whole sarcoglycan complex, with a consequent reduction of sarcolemma fragility. This is a treatment strategy that needs further investigation (73).

LGMD R4 beta-sarcoglycan-related (previously limb-girdle muscular dystrophy 2E) (4q) – beta-sarcoglycan. Cases of autosomal recessive limb-girdle muscular dystrophy among members of the old order Amish of northern and southern Indiana have been known for almost 3 decades (240). Families from northern Indiana were shown to carry calpain-3 mutations discussed above (422). The Amish families from southern Indiana were found to have mutations in the gene coding for beta sarcoglycan (295). Clinical features of beta-sarcoglycan-related limb-girdle muscular dystrophy resemble those of calpain-3-associated limb-girdle muscular dystrophy (limb-girdle muscular dystrophy R1). Both limb-girdle muscular dystrophy R4 and R6 may be associated with cardiomyopathy, probably because of abnormalities of the sarcoglycan complex in vascular smooth muscle. Beta sarcoglycan-deficient mice also demonstrate secondarily leaky ryanodine receptors, a mechanism that may underlie muscle degeneration and might provide a novel therapeutic target (05).

In a multicentric study of 32 patients with LGMD R4, there was proximal muscle weakness in all patients, but distal involvement was also observed in patients with severe disease early in the disease course. The first symptoms occurred before age 10 years in all patients with severe disease and in 4 of 12 patients with mild disease. All patients with severe disease lost independent ambulation at a mean age of 13 years (range 9 to 17 years). Age at loss of ambulation was between 23 and 59 years in five patients with mild disease (mean 40 years). Frequent clinical signs included calf hypertrophy (78% of patients), tiptoe gait pattern (56%), tendon contracture (69%), scapular winging (59%), and scoliosis (59%). Muscle pain was reported in 75% of mild cases, 40% of unknown phenotype, and in no severe cases. No rhabdomyolysis events were reported. Macroglossia was found in 31% of patients, with almost double prevalence in severe versus mild cases. Cardiac involvement was observed in 20 patients (63%) even before overt muscle involvement. Six patients had restrictive respiratory insufficiency requiring assisted ventilation (19%). There is a significant decrease in cardiac and respiratory functions over time. Ejection fraction is the biomarker that most steadily decreases with disease progression. A decrease in Cpk values is seen with disease progression as there is loss of muscle mass (319). Seventeen different mutations were identified, and three were recurrent. The c.377_384dup (13 alleles) was associated with the severe form, the c.-22_10dup (10) with the milder form, and c.341C.T (9) with both. The entire sarcoglycan complex was absent in 9 of 10 severe cases and reduced in 7 of 7 mild cases. The residual amount of sarcoglycan in muscle was a predictor of age at loss of ambulation (457).

In an LGMD R4 mouse model, after the intramuscular or intravenous injection of beta-sarcoglycan gene driven by a muscle-specific tMCK promoter (scAAVrh74.tMCK.hSGCB), 91.2% of muscle fibers in the lower limb expressed beta-sarcoglycan, restoring assembly of the sarcoglycan complex and protecting the membrane from Evans blue dye leakage (407; 408).

LGMD R5 gamma-sarcoglycan-related (previously limb-girdle muscular dystrophy 2C) (13q) – gamma-sarcoglycan (SGCG). Early works failed to distinguish this severe childhood autosomal recessive form of muscular dystrophy (SCARMD) from Duchenne dystrophy (286; 473; 536). The observation that both girls and boys in the same sibship had Duchenne-like dystrophy was clear evidence that SCARMD was a separate entity.

The locus was subsequently identified as the site of the gene coding for gamma sarcoglycan (365). LGMD R5 is the most frequent limb-girdle muscular dystrophy in North African populations as a result of the founder mutation c.525delT (129). The G787A mutation has been described in unrelated children of Puerto Rican ancestry, suggesting a founder effect (113). The first symptoms appear between 3 and 12 years of age. Pelvic girdle weakness precedes pectoral girdle weakness. However, there is considerable variability in the severity of the disease among siblings and between families. Loss of independent ambulation generally occurs in the second or third decade of life (half of the patients lose ambulation by age 12 years). Progressive scoliosis, macroglossia, and reduction of functional vital capacity are also present. Calf hypertrophy and cardiac involvement are frequent in patients from North Africa (36). Other studies suggest that the typical Tunisian mutation (delta 521T) is associated with milder cardiac manifestations whereas more severe phenotypes are seen in association with entire deletions of exon 7 (402). A wide variety of phenotypes have been reported in a large series of patients with the Tunisian 521T deletion, suggesting that other genetic factors influence the phenotype or that the phenotype responds to environmental influences (259). A case with episodic myoglobinuria is also reported, a pattern more common in metabolic myopathies or occasionally dystrophinopathies (393). A case of embolic stroke secondary to dilated cardiomyopathy has been described (153). Murine studies also suggest that the gamma-sarcoglycan mutations can cause varying phenotypes in different genetic backgrounds that suggest modifying loci that alter the dystrophic phenotype (219).

Histological features resemble those in Duchenne dystrophy, except that there are fewer hypercontracted fibers and less interstitial fibrosis. Overexpression of the gamma subunit in transgenic mice results in upregulation of alpha and beta sarcoglycans, suggesting a role for the gamma subunit in the regulation of the complex as a whole (566). Prominent eosinophilic infiltration, mimicking eosinophilic myositis was described in one patient (28). Patients with this form may also show secondary reduction of dystrophin, which complicates the diagnosis and argues for a direct association of dystrophin with the gamma subunit.

Prenatal diagnosis is possible by demonstrating absence of exon 5 of the gamma sarcoglycan gene in the fetus by chorionic villus sampling (117).

A French group has demonstrated tolerance of adenovirus-associated virus gene transfer of gamma sarcoglycan that included active mRNA and persistent gene expression after 30 days in nine patients (217). An AAV2/8-expressing gamma sarcoglycan controlled by a muscle-specific promoter has been shown efficacious in Sgcg−/− mice through intravenous injection. AAV8 serotype was more efficient in the transduction of striated muscles and gamma sarcoglycan was able to reach therapeutic levels with systemic administration (237). In a mouse model of LGMD R5, treatment with fingolimod (FTY720) produced significant functional benefit by plethysmography and significant reductions of membrane permeability and fibrosis. Furthermore, the protocol elevated protein levels of delta-sarcoglycan, a dystrophin-glycoprotein family member. This indicates that fingolimod could be useful for treatment in the future (218). In vitro, the use of morpholino oligomers to cause gamma sarcoglycan gene exons 4, 5, 6, and 7 skipping resulted in the production of a functional mini-Gamma product containing only exons 2, 3, and 8. This strategy could be used in patients with LGMD R5 affected by different mutations (552).

This multiexon skipping approach was demonstrated to be effective in correcting the aberrant reading frame in vivo using multiple exon-directed antisense oligonucleotide cocktail in a mouse model of LGMD R5 with SGCG 521delT (112).

LGMD R6 delta-sarcoglycan-related (previously limb-girdle muscular dystrophy 2F) (5q) – delta-sarcoglycan. The least common of the sarcoglycanopathies is caused by a mutation in the delta sarcoglycan gene. This mutation has been documented frequently in Brazil (125), where there is a probable founder effect as well as a high degree of consanguinity among the parents of patients with limb-girdle muscular dystrophy R6 (562). This is a very severe and quickly progressive disease characterized by generalized muscle weakness (03). Hypermobility of the interphalangeal, metacarpophalangeal, and elbow joints and delayed language development can also be present (540). In a delta sarcoglycan null mouse, prednisolone, hypothesized to improve cardiac function, actually caused further cardiac damage in animals with cardiomyopathy (27).

LGMD R7 Telethonin-related (previously limb-girdle muscular dystrophy 2G) (17q) – Telethonin. This form of limb-girdle muscular dystrophy was localized to the TCAP gene on 17q11-12 coding for telethonin, a sarcomeric protein (347). The protein, which may be important in myofiber assembly, localizes to the Z-band, where it interacts with titin. Mutations in this gene were initially documented in a few Brazilian and subsequently reported in patients originating from China, Moldavia, Portugal, India, Spain, and Turkey. Reported patients had elevated creatine kinase, onset of proximal weakness in early teens, early toe-walking, thigh atrophy, and calf hypertrophy (50% of cases) and were wheelchair-dependent by mid to late 30s. Upper extremities are minimally affected in some cases (61). Facial weakness can be present, especially after long disease duration. Even in patients with predominant proximal lower limb weakness, foot drop or frequent tripping, related to tibialis anterior involvement, are common initial or late findings (10 years after first symptoms) (99). Achilles tendon and other joint contractures, hyperlordosis, and scapular winging are common. Some patients also report myalgias. Cardiac involvement occurs in some patients, and the clinical course seems to be milder in women than in men (562). Muscle biopsy may show rimmed vacuoles or nemaline rods (379). Muscle MRI shows atrophy and fatty infiltration predominantly in glutei, hip, and thigh muscles and tibialis anterior with the typical sparing of the sartorius muscles (233; 88). Diagnosis is suggested by immunohistochemistry against telethonin in muscle tissue where it would be markedly decreased or absent; however, genetic testing is needed to confirm diagnosis as in some mutations telethonin staining of a truncated form can be present (false negative results) (65; 22). TCAP mutations also cause congenital muscular dystrophy and dilated cardiomyopathy. A Chinese study found additional pathogenic TCAP variants for LGMD R7, including an intronic variant, with the most common variant being c.165-166insG (307). The authors also provided evidence for autophagy as part of the pathophysiology for LGMD R7.

LGMD R8 TRIM 32-related (previously limb-girdle muscular dystrophy 2H) (9q) – TRIM32. TRIM32, a ubiquitin ligase, is expressed in skeletal muscle and interacts with myosin, ubiquinates actin, and likely is involved in maintenance and degradation of myofibrils during muscle remodeling (275). It was suggested that TRIM32 is involved in the ubiquitin proteosome pathway, a specialized pathway for post-translational regulation of protein levels (166; 282). The protein is localized to the Z-line in skeletal muscle and appears to regulate dysbindin; LGMD R8 and STM mutations may impair substrate ubiquitination (300). Immunostainings for desmin and myotilin, substrates of the TRIM32 E3 ligase, were seen increased in the muscle fibers of patients with LGMD R8 (384). Thin, a Drosophila protein highly analogous to TRIM32, is critical to myofibril stability. Fly mutations in this gene produce muscular degeneration (279). In particular, costameric integrin and sarcoglycan protein levels are altered in a Drosophila model for LGMD R8 (29). TRIM32 also regulates skeletal muscle stem cell differentiation and is necessary for normal adult muscle regeneration and myogenic cell proliferation and differentiation (363; 344). Mutations in this gene cause seemingly diverse diseases, including limb-girdle muscular dystrophy (LGMD R8), sarcotubular myopathy, and Bardet-Biedl syndrome type 11.

LGMD R8 has been seen in Hutterites of Manitoba (542). Onset is typically in the second to third decade with slow progression. Most patients remain ambulatory into the sixth decade, without cardiac or facial involvement. CK is mildly to moderately elevated. Rare patients can have peripheral neuropathy (TRIM32 is expressed in nerve). Muscle MRI shows predominant involvement of the posterior thigh and leg compartments, with relative sparing of the flexor digitorum longus, flexor hallucis longus, and tibialis posterior muscles (250).

Frosk and colleagues also describes a Hutterite family with two brothers homozygous for both mutations (164). The mother, father, and five sons were homozygous for a TRIM32 mutation and 2 of 5 sons were also homozygous for L276I mutations in FKRP. The double homozygous exhibited mild decrease in stamina with normal strength. The homozygous LGMD R8 (TRIM32)/heterozygous LGMD R9 (FKRP) family members were virtually asymptomatic. Other mutations have also been found. Borg and colleagues reported a large Swedish family with limb girdle muscular weakness and histological features of a microvacuolar myopathy; the index patients were compound heterozygotes. Family members with one mutation had a milder phenotype; demyelinating neuropathy was also seen in some affected family members (53). A patient with a scapuloperoneal phenotype has also been identified (293).

LGMD R9 FKRP-related (previously limb-girdle muscular dystrophy 2I) (19q) – Fukutin-related protein. Fukutin-related protein gene (FKRP) had been shown to be mutated in severe forms of congenital muscular dystrophy (MDC1C). FKRP gene mutations have also been demonstrated in patients with limb-girdle muscular dystrophy R9, making limb-girdle muscular dystrophy 2I and MDC1C are allelic disorders. FKRP and Fukutin, a protein mutated in Fukuyama congenital muscular dystrophy, share sequence similarities with proteins involved in modifying cell surfaces. FKRP encodes a glycosyltransferase in the Golgi apparatus that is involved in alpha-dystroglycan glycosylation. FKRP transfers ribitol-5-phosphate to alpha-dystroglycan from cytidine diphosphate-ribitol, which is synthesized by isoprenoid synthase domain-containing protein (ISPD) (254). Dimer form of FKRP is important for substrate recognition and its enzymatic activity. Mutations that cause deficiency in oligomerization or enzymatic function of FKRP may lead to dystroglycanopathy (215; 278). There is also some evidence of metabolic impairments in FKRP-deficient skeletal muscles (516). Downregulating FKRP expression in zebrafish by two different morpholinos resulted in embryos that had developmental defects and glycosylation deficiency similar to those observed in human muscular dystrophies associated with mutations in FKRP (256). In this model, co-injection of fish or human FKRP mRNA restored normal development, alpha-dystroglycan glycosylation, and laminin-binding activity of alpha-dystroglycan in the morphants. Mutations in either protein result in secondary lamin alpha 2 and alpha dystroglycan deficiency (57). Moreover, there appears to be a correlation between residual alpha-dystroglycan expression and phenotype. However, in the zebrafish model, FKRP-associated dystroglycanopathy does not completely phenocopy dystroglycan-deficiency (16). A study showed that there was an elevation of autophagy markers and downregulation of a negative autophagy regulator mTORC1 in LGMD R9, indicating an activation of autophagy (163). There was also an increased expression of endoplasmic reticulum stress markers.

Brown and colleagues have identified three broad categories of human FKRP disorders:

(1) Severe congenital patients (MDC1C) were found to be compound heterozygotes with either a null allele and missense mutation or two missense mutations and correspondingly profound alpha-dystroglycan depletion (59).

(2) Patients with limb-girdle muscular dystrophy with a Duchenne-like severity typically had moderate alpha-dystroglycan reduction and were compound heterozygotes for a common FKRP mutation L276I (c.826C> A) and either a missense or a nonsense mutation. Schwartz and colleagues screened samples for the L276I mutation from sporadic, male patients who were referred for dystrophinopathy and had no dystrophin deletions (454). Thirteen cases were found, all with the L276I point mutation. All patients had CK levels ranging from 1000 to 5000 and calf hypertrophy. About one third had loss of ambulation by age 36 and others had cardiac and respiratory deficits. A man who had originally been diagnosed with Duchenne muscular dystrophy based on his phenotype was discovered to actually have LGMD R9, with genes heterozygous for FKRP variants: c.169G>A (p.Glu57Lys) and c.692G>A (p.Trp231*) (371).

(3) Individuals with a milder form of limb-girdle muscular dystrophy 2I were almost invariably homozygous for the common L276I (c826C> A) FKRP mutation and showed a variable but subtle alteration in alpha-dystroglycan immunolabeling. A database review of these patients found that they tended to have a better prognosis in terms of age of diagnosis as well as milder symptoms in comparison to the other subgroups of FKRP mutations, although they still had high rates of cardiac involvement and many still required assistive ventilation at some point (358).

Although variable phenotypic severity has been partly attributed to the differences in the mutations, there has been significant phenotypic variation reported among individuals with the same FKRP mutation (472). The modifiers are unknown. Mouse models of FKRP mutations also show a wide range of disease phenotypes (47).

There have been founder mutations in FKRP reported in European (c.826C> A, LGMD R9), Tunisian (c.1364C> A, congenital muscular dystrophy with brain involvement), Chinese (c. 545A> G, asymptomatic), South African Afrikaner (c.1100C> T, LGMD R9), and Mexican (c.1387A> G congenital muscular dystrophy without brain involvement) populations (304; 165; 167; 351; 283).

LGMD R9 was originally described in a large consanguineous Tunisian family and linked to 19q (122). Poppe and colleagues described the clinical features of limb-girdle muscular dystrophy R9 patients, most of whom had the same C826A mutation. The phenotype included calf hypertrophy, marked creatine kinase elevation, and frequent cardiac and respiratory involvement (405). Over half had cardiac abnormalities, and half of these developed heart failure (404). Forced vital capacity was below 75% in 44% of patients. Oddly, heterozygotes developed cardiac involvement earlier than homozygotes. Isolated dilated cardiomyopathy without limb weakness has been reported in three siblings who were homozygous for the L276I (c826C> A) mutation (354).

The most common FKRP mutation, L276I (c826C> A) in the Hutterite families discussed earlier, is also found in many non-Hutterite families in multiple European countries, Canada, and Brazil, suggesting a founder effect dispersed throughout populations of European origin (165). It is thought to be the most common form of autosomal recessive limb-girdle muscular dystrophy in northern Europe, especially in Denmark, with a reported 38% prevalence of LGMD R9 in the adult recessively inherited limb-girdle muscular dystrophy population, which is a 3- to 4-fold higher prevalence than in other regions (485).

A different mutation in FKRP, A455D, was identified in Tunisian patients with a severe MDC1C phenotype and mental retardation, microcephaly, and cerebellar abnormalities (304). Another phenotype presents as an acute myositis, mimicking viral myositis in infants (531). Of clinical importance, diaphragmatic involvement may cause respiratory insufficiency in patients who remain ambulant (65). Two patients with severe congestive heart failure (severe enough to require cardiac transplantation) but mild muscular findings have been described (320).

Boito and colleagues screened 214 Italian patients with muscular dystrophy and normal dystrophin, alpha-sarcoglycan, calpain-3, and dysferlin immunohistochemistry; FKRP mutations were identified in 6% of patients (13/214) and about one third of these patients had the L276I (c.826C> A) mutation (49). Patients exhibited phenotypic variability within families and among the entire cohort. One homozygote and another heterozygote were completely asymptomatic whereas others had severe cardiac and pulmonary disease. Most patients had predominant hip girdle weakness and calf hypertrophy. In a reported family, some heterozygous carriers of the c.826C> A mutation showed symptoms including calf pseudohypertrophy, scapular winging, and cardiomyopathy (451). Myalgias are also common. A case of LGMD2I and unilateral cataracts (556) and metacarpophalangeal joint hypermobility have been described 539).

Mutations located in the intron of FKRP may also be pathogenic. A case of early-onset LGMD R9 with compound heterozygous c.-253+4A>G and c.826C> A has been reported (445). Willis and colleagues described a novel mutation of single nucleotide insertion (c.948_949insC) and an 18-nucleotide duplication (c.999_1017dup18) in FKRP associated with LGMD R9 (546). Muscle MRI shows that the glutei muscles, adductors, biceps femoris, vastus intermedius, and vastus lateralis are more affected. The rectus femoris, sartorius, and gracilis are relatively spared (540; 553). Cases with only mild adductor magnus fatty infiltration also exist (540). Fat fraction measurement using 3‐point Dixon MRI technique has been shown to be a sensitive marker of disease progression and may be used as a primary outcome measure alongside functional assessments in clinical trials in the future (357).

In a study of nine patients with LGMD R9 after 50 cycling sessions of 30 minutes each over a 12-week period (achieving 65% of their maximal oxygen uptake), the patients had improved exercise capacity and showed no significant increase in CK levels or altered muscle morphology, supporting the benefit of moderate-intensity endurance training in this particular phenotype (484).

In a study of eight patients with LGMD R9, all patients showed abnormal ON/OFF bipolar cell responses and sawtooth 30 Hz flicker waveforms on full-field electroretinogram (202). Further studies are needed to confirm whether this is a specific finding for LGMD R9.

Treatment with prednisolone has been described in two patients with a Duchenne type presentation; both had good response (107). Another patient with necrotizing myopathy in muscle biopsy showed response to steroids, azathioprine, and intravenous immunoglobulin (482). A single-dose treatment of AAV9-FKRP in FKR PP448L mutated mice after disease onset significantly improved the pathology and functional activity over an extended period of time and was even able to extend the lifespan of mice in advanced stages of disease progression (517). In a mouse model homozygous for the P448L mutation that presents as congenital muscular dystrophy, the oral administration of ribitol restored therapeutic levels of functional alpha dystroglycan in skeletal and cardiac muscles. Furthermore, ribitol, given before and after the onset of disease phenotype, reduced skeletal muscle pathology, decreased cardiac fibrosis, and improved skeletal and respiratory function. Ribitol could potentially be used for other FKRP mutations (78).

ISPD overexpression alone and in combination with ribitol has also been shown to improve dystrophic phenotype in a FKRP mutant mouse model (77). Mouse myoblast line and human iPSCs derived from patients with FKRP mutations have been used to screen for compounds that significantly augmented glycosylation of α-dystroglycan and may facilitate drug development for dystroglycanopathies (263).

LGMD R10 titin-related (previously limb-girdle muscular dystrophy 2J) (2q)--titin. Tibial muscular dystrophy, an autosomal dominant adult-onset distal myopathy, was described in a large Finnish pedigree and associated with defects in the protein titin (201). Titin, the biggest single peptide in humans (greater than 38 KDa), stretches from the M-line to the Z-line. Titin has mechanical, developmental, and regulatory functions in striated muscle, and has multiple ligand binding sites, including calpain-3, telethonin, and alpha-actinin (201). Titin is thought to stabilize calpain-3 and protect it from autolytic degradation; in fact, some patients with tibial muscular dystrophy had secondary calpain-3 deficiency (206), supporting the interaction between titin and calpain-3 discussed earlier. Allelic titin disorders include dilated cardiomyopathy without skeletal muscle disease, arrhythmogenic right ventricular cardiomyopathy (ARVC) and monogenic restrictive cardiomyopathy (RCM), familial hypertrophic cardiomyopathy, early onset myopathy with fatal cardiomyopathy (EOMFC), hereditary myopathy with early respiratory failure (HMERF), childhood-onset Emery Dreiffus-like phenotype without cardiomyopathy, multiminicore disease with heart disease, congenital centronuclear myopathy, and young- or early adult-onset recessive distal myopathy.

A mouse model with muscular dystrophy and myositis also shows a deletion in the titin gene likely at the calpain-3 binding site and may be a useful animal model (176). Targeted deletion of the C-terminal end of titin, which contains a kinase domain that phosphorylates telethonin, results in impaired myofibrillogenesis in organizing muscle sarcomeres (336).

Heterozygotes (autosomal dominant) have late onset, slowly progressive distal myopathy, especially of the anterior compartment, and lesser proximal weakness. Cardiac involvement has not been described.

A single titin mutation, Finnish FINmaj, is an 11bp insertion-deletion mutation exchanging four amino acids in the 363rd and last exon of TTN (Mex6), which encodes the C-terminal immunoglobulin domain M10 of M-line titin. It was found in 211 of 386 affected Finnish patients, and there was considerable phenotypic variation (506). The vast majority (91%) had the tibial muscular dystrophy phenotype consisting of late-onset (older than 35 years of age), slowly progressive distal myopathy, especially in the anterior compartment, and lesser proximal weakness with sparing of the short toe extensor digitorum brevis muscle. Nine percent had atypical presentations that included proximal weakness, bulbar weakness, asymmetric weakness, and childhood-onset weakness. Biopsy findings include fiber size variability, central nuclei, necrosis, presence of fibroadipose tissue and rimmed vacuoles. Electron microscopy showed autophagic vacuoles without membrane and very rare inclusions of 15 to 18 nm filaments. Muscle imaging (CT or MRI) is very informative with selective fatty replacement in the muscles of the anterior compartments of the lower legs starting in the anterior tibial muscle and represents a useful clinical tool to address the diagnosis (444). In congenital titinopathies, muscle MRI usually shows fatty infiltration or atrophy at paraspinal, gluteal, and hamstring muscles, whereas the adductors, sartorius, and gracilis are usually spared or hypertrophied (367; 560).

In the original Finnish pedigree, a few patients were affected in childhood with more severe limb-girdle muscular dystrophy and were found to be homozygous for titin mutations; parents of these homozygotes were not affected (206). The homozygous phenotype was adopted as autosomal recessive LGMD R10 (562). Only 2 of 211 patients were homozygous for FINmaj, and neither of these had the tibial muscular dystrophy phenotype. LGMD R10 is a severe childhood onset disease causing proximal muscle weakness in the first or second decade and progresses over the next 20 years to wheelchair confinement. Because the TTN mutation affects the A band, cardiac problems may occur first or may overlap with or follow skeletal muscle weakness (421). LGMD R10 shows a secondary CAPN3 defect. Most biopsied muscles of patients homozygous for the FINmaj variant show dystrophic findings with end-stage pathology without rimmed vacuoles although a case with rimmed vacuoles has also been reported.

A study of muscle biopsy in patients with different titin mutations found that findings depend on the phenotype. Autosomal recessive congenital myopathy showed defects in oxidative staining with prominent nuclear internalization. Autosomal recessive Emery-Dreifuss-like and autosomal recessive adult-onset distal myopathy phenotypes showed necrotic/regenerative fibers associated with endomysial fibrosis and rimmed vacuoles. Hereditary myopathy with early respiratory failure showed cytoplasmic bodies as a predominant finding (13).

A French family with an autosomal-dominant, late-onset distal myopathy of the tibial muscular dystrophy phenotype has been described (394). One deceased patient in the family proved to be homozygous for the C-terminal truncating titin mutation because of consanguinity. A patient presenting with inflammatory infiltrates in muscle biopsy resembling polymyositis refractory to different immunosuppressants has also been described (104).

Measurement of urinary N-terminal fragment of titin/creatinine ratio has been shown to have good 100% sensitivity and 92% specificity in diagnosing cardiomyopathy secondary to muscular dystrophies versus other etiologies (558).

Diagnosis of titinopathy can be difficult because genetic testing can find different variants that may not be causing disease. Savarese and colleagues proposed a workflow for interpreting titin variants (443). If a previously reported, disease-causing mutation is found, the diagnosis is made. Identifying two truncating variants on both the alleles results in a diagnosis of titinopathy. A single heterozygous protein truncating variant is not sufficient for a diagnosis of titinopathy. Missense variants can lead to a diagnosis of titinopathy only when there is sufficient evidence supporting their pathogenicity (functional studies, Western blot, etc.) (443).

LGMD R11 POMT1-related (previously limb-girdle muscular dystrophy 2K) (9q) – POMT1. POMT1 along with POMT2 gene products act to transfer O-mannosyl glycan chains onto alpha-dystroglycan.

An autosomal recessive limb-girdle muscular dystrophy with mental retardation was reported in six Turkish families (116). The consensus phenotype in the eight published cases was mild proximal weakness starting early (age of onset ranged from 1 to 6 years old), mild calves or thigh pseudohypertrophy, elevated CK (range 9 to 40 times normal), microcephaly (range of head circumference 3rd to 50th percentile), and mental retardation (range of IQ 50 to 76). Other than microcephaly, neuroimaging studies were normal. Immunohistochemical analysis of muscle biopsy samples revealed decreased alpha-dystroglycan expression. In five of the Turkish patients (116), Balci and colleagues (18) identified the culprit gene as POMT1 (O-mannosyltransferase), the same gene involved in 7% to 20% of patients with Walker-Warburg syndrome (35; 102) and congenital muscular dystrophy with mental retardation. Haberlova reported two sisters with POMT1 mutations in whom psychiatric symptoms including psychosis preceded the onset of limb girdle weakness (198).

Walker-Warburg syndrome is more severe than LGMD R11 and is characterized by severe cerebral malformations leading to type II lissencephaly, cerebellar and pontine hypoplasia, hydrocephalus, and congenital muscular dystrophy. Another allelic variant presents as severe motor impairment, leg hypertrophy, and mental retardation without brain or eye malformations (103). A dermal fibroblast-based assay of enzyme activity on the glycosylation status of alpha-dystroglycan, protein O-mannosyltransferase activity, and the stability of the mutant POMT1 protein was directly correlated with the severity of the clinical phenotype and inversely correlated with POMT activity (303).

To date, more than 76 disease-associated POMT1 mutations have been reported in the literature (228). Geis and colleagues described 35 POMT1 patients (including eight previously published cases) from 27 independent families of various ethnic origins. Fifteen patients had Walker-Warburg syndrome, one patient had muscle-eye-brain disease-like phenotype, and 19 patients were categorized as LGMD with mental retardation phenotype. There is genotype-phenotype correlation of POMT1-related disorders. Biallelic mutations leading to premature protein truncation result in a severe Walker-Warburg syndrome phenotype. A missense mutation might result in a milder phenotype if not located in a protein domain essential for the catalytic enzyme activity (178).

LGMD R12 Anoctamin5-related (previously limb-girdle muscular dystrophy 2L) (11p) – ANO5. ANO5 is a putative calcium-activated chloride channel and is involved in muscle membrane function and repair (190), but its function is not completely understood (297). ANO5 mutations have been discovered to cause defective annexin coordination during cellular repair (161). Mouse models have also demonstrated that ANO5 mutations cause cytosolic calcium overload with plasma membrane injury, which compromises muscle fiber repair (84). It is expressed in human bone and cardiac and skeletal muscles. Defects in this gene are also associated with the rare skeletal disorder gnathodiaphyseal dysplasia. Despite the relatively late discovery of this gene, it may be more common than others; ANO5-associated limb-girdle muscular dystrophy is reported to be the third most common type of limb-girdle muscular dystrophy in Northern and central Europe, possibly because of a founder effect. This gene was found to be 2% of an Italian cohort of limb-girdle muscular dystrophy and Miyoshi-like patients (220; 312). A Finnish group found eight different mutations in 25% of a varied cohort that included 75% Finnish and varied other origin patients (396). The most common ANO5 variants are c.191dupA in exon 5 and c.2272C>T in exon 20. Testing of these two exons is sufficient for molecular diagnosis in a majority of cases, particularly in individuals of northern or central European descent. LGMD2L was relatively rare among Asians. No recurrent mutation has been identified in Asian populations so far (69).

Anoctamin 5 (ANO5) mutations can present as LGMD R12 (LGMD2L) (50), Miyoshi-like distal myopathy type 3, asymptomatic hyperCKemia, and myoglobinuria after exercise (385).

Asymmetrical weakness and atrophy of quadriceps, biceps brachii and calf muscles as well as myalgia and elevated serum creatine kinase (CK) levels are common clinical features of anoctaminopathy. Some patients have calf hypertrophy (227). Onset of weakness can vary from the second to seventh decade (average 35 years of age). Female patients seem to have a milder phenotype than male patients (294), although early onset of weakness was reported in a woman who did heavy exercise training, which may have caused more sarcolemmal membrane injury (46). Jarry and colleagues described 14 patients from eight different families with asymmetric quadriceps femoris atrophy that evolved into a limb-girdle muscular dystrophy phenotype (244). Creatine kinase levels ranged from normal to 6000; age of presentation ranged from less than 20 to 60 years of age, but average age of onset is around 30 years. Phenotypes varied among individual family members and across different families. Manifesting ANO5 mutation carriers with mildly elevated CK, myalgias, and without muscle weakness have also been described (253). Some patients are also asymptomatic despite elevated CK levels (447). Patients with hypertrophic or dilated cardiomyopathy of possible association have been reported. Cardiac arrhythmia, including bradycardia, paroxysmal atrial fibrillation, and paroxysmal supraventricular tachycardia, is also present in some patients (512). Possible association with macular dystrophy in one patient has also been postulated because ANO5 transcripts have been identified in the retinal pigment epithelium, choroid, and fetal eye (519).

Muscle MRI may show asymmetric muscle involvement, but the pattern was not found to distinguish this phenotype from others such as Bethlem myopathy (438). Predominant involvement of the gluteus minimus muscle and both the posterior segment muscles of the thigh and calf with relative sparing of the gracilis muscle similar to dysferlin mutation is seen (497). A reported patient had isolated semitendinosus involvement in MRI (491). EMG can show electrical fibrillations, positive sharp waves, myopathic motor units, and electrical myotonia without clinical myotonia (294). Muscle biopsy can show rare rimmed vacuoles and also amyloid deposition (ANO5 is not present in those deposits) in the walls of the intramuscular blood vessels and in the endomysium and often surrounding perifascicular muscle fibers. A case with biopsy findings of necrotizing myopathy unresponsive to immunosuppression has also been reported (449). ANO5 mutation-related myopathy is the second most common cause of amyloid myopathy after immunoglobulin light-chain amyloidosis (292). There is no difference in the mutation location or the clinical presentation between patients with intramuscular interstitial amyloid deposits (53% of cases) and those without that finding (04).

In six patients with LGMD R12, home-based, pulse-watch monitored, moderate-intensity exercise on a cycle ergometer for 30 minutes, 3 times weekly for 10 weeks resulted in improvements in maximum oxygen uptake (VO2max) and time in the five-repetitions-sit-to-stand test (FRSTST) without any changes in CK levels (527).

LGMD R13 Fukutin-related (previously limb-girdle muscular dystrophy 2M) (9q) – Fukutin (FKTN). Three patients (Israeli and Indian origin) from two nonconsanguineous families demonstrating abnormal alpha-dystroglycan on muscle biopsy were studied. The patients’ weakness began at less than 1 year of age. Legs were affected more than arms, and calf hypertrophy was seen. CKs ranged from 9000 to 60,000 with inflammatory features seen on biopsy suggestive of polymyositis. All three patients had episodes of deterioration with febrile illnesses. Steroids led to improvement and subsequent stabilization. All patients were able to walk. Mutations were found in the Fukutin gene, the same gene that causes Fukuyama congenital muscular dystrophy (182). Fukutin acts as a ribitol phosphate transferase in the Golgi apparatus to catalyze the biosynthesis of tandem ribitol phosphate structures on alpha-dystroglycan by sequential action with fukutin-related protein (254). Reduced glycosylation on alpha-dystroglycan may disrupt the link between the intracellular cytoskeleton to the extracellular basement membrane through the dystrophin-glycoprotein complex, affecting cardiac muscles, skeletal muscles, and brain (508; 504).

Onset of weakness in LGMD R13 is usually between 4 months to 7 years (although a patient with late onset at 14 years of age has been reported). Calf hypertrophy, lumbar lordosis, joint contractures, scapular winging, and rigid spine can be seen in these patients. A patient with Wolff-Parkinson-White syndrome and LGMD R13 has been described (424). The CK values decreased with steroid treatment in one patient (466).

Fukuyama congenital muscular dystrophy, described in 1960, presents as generalized weakness, severe brain damage, epilepsy, and abnormal eye structure or function and is the second most common muscular dystrophy in Japan after Duchenne muscular dystrophy (168). Another allelic variant presents as dilated cardiomyopathy with little or no muscle involvement (356). Defects in this gene are a recognized cause of childhood onset muscular dystrophy without mental retardation (411) and can also present with asymptomatic hyperCKemia (159).

LGMD R14 POMT2-related (previously limb-girdle muscular dystrophy 2N) (14q) – POMT2. Protein O-mannosyltransferase 2 (POMT2) is a protein involved in alpha dystroglycan glycosylation. Mutations in POMT2 have been identified in patients with congenital muscular dystrophy and brain involvement, either characterized by a Walker-Warburg or muscle-eye-brain phenotype or by microcephaly, mental retardation, and cerebellar hypoplasia. In 2007, Biancheri reported a 5-year-old girl with POMT2 mutation and limb-girdle muscular dystrophy phenotype, calf hypertrophy, elevated CK (3350), and macrophage inflammation in muscle biopsy. She received steroids with resultant reduction in CK levels. She had no mental retardation, eye problems, or brain MRI abnormality (43).

A case series of 12 patients provides a better description of LGMD R14 (LGMD2N) manifestations; onset varied from birth to age 55 years. Patients with birth onset still walked unassisted at 18 years of age. Symptoms at onset include walking difficulty, learning impairment, and myalgias. Four patients had reduced Mini Mental Exam (MMSE) scores. One patient had dilated cardiomyopathy, and other had decreased left ventricular ejection fraction. Functional vital capacity was reduced in all patients at an average of 66%. Hip and knee flexors and extensors were the weakest. Calf hypertrophy was seen in some patients. Scapular winging was seen in three patients. CK was elevated (from 398 to 5000). Brain MRIs were abnormal in 3 of the 10 patients in whom brain imaging was carried out, showing mild ventricular enlargement due to central and cortical atrophy, periventricular hyperintensities, and frontal atrophy of the left hemisphere. Muscle MRI revealed a pattern of selective muscle involvement, most strikingly affecting the hamstring (worst), paraspinal, and gluteal muscles. In the leg, the posterior compartment muscles were more affected (376).

Three siblings with congenital muscular dystrophy caused by POMT2 mutations, significant dilatation of the aortic root, and depressed left ventricular systolic function or left ventricular wall motion abnormalities have been described. For this reason, frequent cardiac surveillance is recommended in patients with POMT2 mutations (321). Other features that have been described in patients with POMT2 mutations include polycystic kidneys, atrial ventricular septal defects, left ventricular hypertrophy, ventricular arrhythmias, scoliosis, café-au-lait spots, axillary freckling, and skin hyperpigmentation (158).

Muscle biopsy shows dystrophic changes with selective reduction of alpha-dystroglycan immunostaining (60).

LGMD R15 POMGnT1-related (previously limb-girdle muscular dystrophy 2O) (1p) – POMGnT1. Protein-O-mannose-_1,2-Nacetylglucosaminyltransferase 1 (POMGnT1) is a glycosyltransferase gene involved in the glycosylation of dystroglycan and is responsible for the transfer of N-acetylglucosamine (GlcNAc) to mannose (318; 222). Mutations have been described in patients with muscle-eye-brain disease, Walker-Warburg syndrome, retinitis pigmentosa 76, and LGMD R15. Clement and colleagues described a girl with onset of proximal upper and lower extremity weakness at the age of 12 years, with calf hypertrophy and lumbar hyperlordosis. She had myopia and surgery to correct convergent squint. Her CK level was elevated (up to 12,000). Muscle biopsy specimen exhibited dystrophia with abnormal variation in fiber size, necrosis, increased endomysial connective tissue and fat, and basophilic fibers, some of which were granular and had vacuoles (94).

Raducu and colleagues described a patient with delayed motor milestones, hypotonia, generalized amyotrophy, lumbar hyperlordosis, and mild CK elevation. Brain MRI and eye examination were normal (414).

LGMD R16 Alpha-dystroglycan-related (previously limb-girdle muscular dystrophy 2P) (3p) – Dystroglycan 1. Dystroglycan is a central component of the dystrophin-glycoprotein complex, which links the cytoskeleton and extracellular matrix through sarcolemma. Dystroglycan has important roles in the development and maintenance of skeletal muscle, the CNS, and other organs. DAG1 mutations cause primary dystroglycanopathy in limb-girdle muscular dystrophy and muscle-eye-brain disease. In 2003, Dincer and colleagues reported a girl with limb-girdle muscular dystrophy and mental retardation. The gene was identified in 2011. Symptoms included progressive proximal muscle weakness at age 3 years, mild calf enlargement, lumbar hyperlordosis, and ankle contractures. Her intellectual development was slow, and she said her first few words at 7 years; at 16 years of age, she only used 2-word sentences. CK was elevated (4000s). Brain MRI was normal (116; 204).

A mild, late-onset limb-girdle muscular dystrophy presentation is also possible (105). A boy with DAG1 mutations and asymptomatic hyperCKemia, calf pseudohypertrophy, and mild dystrophic changes in muscles has also been described (119).

DAG1 mutations are also associated to congenital muscular dystrophy (dystroglycanopathy) with Walker-Warburg syndrome with tectocerebellar dysraphia (285).

LGMD R17 Plectin-related (previously limb-girdle muscular dystrophy 2Q (8q) – Plectin (PLEC). Plectin is a large intermediate filament-binding protein that helps maintain cytoskeleton stability and neuromuscular junction integrity (549). PLEC mutations can cause LGMD R17, different types of epidermolysis bullosa simplex, and congenital myasthenic syndrome (549; 185). Plectin isoform 1f is associated with the sarcolemma whereas isoform 1a is associated with keratin intermediate filaments in basal keratinocytes. Tissue-specific expression and isoform-dependent deficiency of plectin may explain the various phenotypes (420).

There are reports of patients showing myasthenic symptoms as well as epidermolysis bullosa simplex with muscular dystrophy, which are called EBS-MDMys. Regarding limb-girdle muscular dystrophy, Turkish patients with muscular dystrophy but no skin changes were found to have a deletion in this gene (194). Early onset disease that later plateaued was characteristic. Other patients without skin involvement and with limb-girdle muscular dystrophy pattern of weakness, bilateral eyelid ptosis, diplopia, facial weakness, and elevated CK have also been described. Single-fiber EMG was normal, and there was no response to pyridostigmine (152). Pyloric atresia and cardiomyopathy have also been associated with PLEC mutations. Other features include alopecia, onychodystrophy, abnormal dentition, laryngeal webs, respiratory complications, urethral strictures, and mild bilateral hydronephrosis (10; 510).

Togawa and colleagues found heterozygous variants in PLEC gene (c.2668C> T, p.R890C) and ST3GAL6 gene (c.421G> C, p.V141L) in a family with atypical familial amyotrophic lateral sclerosis with slowly progressing lower extremities-predominant late-onset muscular weakness and atrophy. It is unclear whether these variants are pathogenic (500).

Some Turkish women were identified with a c.1_9del mutation in the PLEC gene, with a presentation somewhat similar to a myasthenic syndrome characterized by progressive limb-girdle weakness, muscle biopsy dystrophy, ptosis, facial weakness, fatiguability, and muscle cramps (349). They had clinical improvement with pyridostigmine and salbutamol and also had a common 3.8 Mb haplotype.

LGMD R18 TRAPPC11-related (previously limb-girdle muscular dystrophy 2S) (4q) – trafficking protein particle complex, subunit 11 (TRAPPC11). Transport protein particle (TRAPP) is a multiprotein complex involved in endoplasmic reticulum-to-Golgi trafficking and possibly other membrane trafficking steps. It also plays a role in autophagy (335; 469). TRAPPC11 mutations are associated with congenital muscular dystrophy with fatty liver and infantile onset cataract and also myopathy and intellectual disability, including cerebral atrophy, scoliosis, achalasia, and alacrima (a form of triple A syndrome) (270).

LGMD presentation was described in eight Syrian and Hutterite patients with progressive weakness that started in school age. CK was elevated (9- to 16-fold). There was moderate intellectual disability, infantile hyperkinetic movements, and ataxia. In all affected individuals, the shoulder girdle muscles are less severely involved than the hip girdle musculature. Skeletal findings, present in all affected individuals, included hip dysplasia and scoliosis. Microcephaly, mild bilateral cataract, strabismus convergens, and slight enlargement of the right cardiac ventricle were present in some patients. Respiratory muscle weakness was seen in one patient. Other patients with TRAPPC11 muscular dystrophy have been described with associated retinopathy and natural killer cell dysfunction (281).

Brain MRI showed mild cerebral atrophy in two patients (48; 155) and multifocal diffusion abnormalities in other cases (281). Muscle MRI shows preferential involvement of the posterior leg compartment (281).

LGMD R19 GMPPB-related (previously limb-girdle muscular dystrophy 2T) (3p) – GMPPB. GDP-mannose pyrophosphorylase B (GMPPB) catalyzes the formation of GDP-mannose, which is required for the glycosylation of lipids and proteins. Mutations in GMPPB can lead to hypoglycosylation of alpha‐dystroglycan (75). GMPPB c.803T>C and GMPPB c.1060G>A, associated with LGMD 2T, were found to form cytoplasmic aggregates and were degraded by autophagy-lysosome pathway. These mutations cause abnormal GMPPB distribution and reduced expression (499).

In eight pediatric patients with a dystroglycanopathy due to mutations in GMPPB, all of them presented by 4 years of age. The phenotype ranged from severe congenital muscular dystrophy to limb-girdle muscular dystrophy. Common associated features were hypotonia, epilepsy (40%), intellectual disability (80%), cataracts (40%) and cardiomyopathy (25%); in 30% of patients, brain MRI showed cerebellar hypoplasia (75). Some patients present with congenital myasthenic syndromes with decrement of motor response with 3 Hz nerve stimulation and positive response to pyridostigmine (306; 346).

Other patients have proximal weakness, elevated CK (greater than 17,000), hypertrophic calves, some of them with cardiac involvement including right bundle branch block, sinoatrial block, long QT interval, left ventricular dilatation, and aberrant ventricular conduction. Loss of ambulation was seen by the sixth to seventh decade. Some of them had scapular winging and restrictive respiratory pattern. Episodic rhabdomyolysis as the only symptom was also reported (67). Asymptomatic hyperCKemia and pseudometabolic myopathy or exercise intolerance have also been described (245; 383). Late onset cases (30 to 40 years of age) without brain involvement and with slow progression are also present (19). Patients with GMPPB mutations can have centronuclear myopathy on biopsy and combined pre- and post-synaptic defects of neuromuscular transmission on EMG (364).

Muscle MRI in patients with LGMD R19 shows consistent findings of a preferential affection of paraspinal and hamstring muscles (368).

A case report of a child with GMPPB muscular dystrophy treated with oral prednisone for 3 months showed improvement in muscle strength and function scores and decrease in CK value (154). This could be a future therapy option.

LGMD R20 ISPD-related (previously limb-girdle muscular dystrophy 2U) (7p) – ISPD. ISPD, a gene located on chromosome 7p21, encodes the isoprenoid synthase domain-containing protein and has been implicated in the initial step of the O-mannosylation of alpha dystroglycan. Mutations in this gene have been associated to Walker-Warburg syndrome and muscle-eye-brain disease. ISPD mutations have also been associated with limb-girdle muscular dystrophy of childhood onset with or without cerebellar involvement, white matter changes, or mental retardation. In some cases, patients had calf and tongue hypertrophy, oculomotor apraxia, strabismus, CK elevation, decreased respiratory function, and cardiac ejection fraction (93; 493).

LGMD R21 POGLUT1-related (previously limb-girdle muscular dystrophy 2Z) (3q) – POGLUT1. POGLUT1 (protein O-glucosyltransferase 1), an enzyme involved in Notch posttranslational modification and function, is important for satellite cell regeneration. Four of five siblings from a Spanish family presented with limb-girdle muscular dystrophy. They exhibited muscle weakness predominantly in the proximal lower limbs, with onset during the third decade. The disease course was progressive, leading to scapular winging and wheelchair confinement. Serum creatine kinase level was normal in three patients and mildly elevated in 1. Muscle biopsies from all four affected siblings revealed histological features ranging from very mild myopathic changes to classic dystrophic pathology. Muscle MRI of the legs revealed a striking pattern of muscle involvement, with early fatty replacement of internal regions of thigh muscles that spared the external area (459). Later, Servian-Morilla and colleagues reported a cohort of 15 patients with LGMD R21, from nine unrelated families coming from different countries (458). Age of onset age varied from adult to congenital and infantile onset. Muscle imaging was consistent with previously described "inside-to-outside" fatty degeneration. Patients’ muscle biopsies showed decreased level of the NOTCH1 intracellular domain, reduction of satellite cells, and alpha dystroglycan hypoglycosylation. Their in vitro and in vivo experiments showed POGLUT1 mutations lead to reduced enzymatic activity and/or protein stability. Myogenic activities from transgenic Drosophila and satellite cell-derived myoblasts from patients' muscle samples were reduced. Therefore, muscular dystrophy in patients with LGMD R21 is likely caused by alterations in satellite cell biology from reduced Notch1 signaling, with possibly additional contribution from α-dystroglycan hypoglycosylation (458).

POGLUT1 mutations are also associated with Dowling-Degos disease, an autosomal-dominant hereditary pigmentation disorder that causes varying degrees of progressive reticulate hyperpigmentation, primarily affecting the flexures, large skin folds, trunk, face, and extremities (415). Galli-Galli disease, an acantholytic variant of Dowling-Degos disease, has also been reported with POGLUT1 mutations (272).

LGMD R22 Collagen6-related (previously Bethlem myopathy recessive) (21q, 2q) – collagen type VI subunits alpha-1, 2, and 3. In 1976, Bethlem and Van Wijngaarden described an autosomal dominant, early-onset, but relatively benign limb-girdle muscular dystrophy associated with flexion contractures in many members of three unrelated Dutch families. The progression was extremely slow, with periods of arrest for several decades; the ability to walk was preserved until old age. Ankle and elbow joints seemed preferentially affected and long finger flexor contractures were usually the first signs. Serum creatine kinase was normal or slightly elevated, EMG showed a myopathic pattern, and muscle biopsy disclosed nonspecific myopathic alterations. The defect was mapped to three genes encoding different subunits of collagen type VI. Two of them are on chromosome 21q (alpha-1, alpha-2) and one on chromosome 2q (alpha-3) (248). Onset is earlier than in typical limb-girdle muscular dystrophy; nearly all patients demonstrate weakness or contractures before 2 years of age, but progression is slow and continues into adulthood (247). More than two thirds of patients over 50 years of age are wheelchair bound (247).

An allelic recessive disorder exists—Ullrich scleroatonic muscular dystrophy. This rare autosomal recessive form of congenital muscular dystrophy with distinctive proximal joint contractures, distal hyperextensibility, and normal intelligence has been associated with different mutations in collagen VI subunits (221; 515). The use of skin biopsy with COL6 staining is postulated to facilitate diagnosis (82).

The autosomal dominant form of collagen VI-related dystrophies is designated as LGMD D5 as discussed above.

Interestingly, mutations in COL6A3 have also been associated with a form of isolated recessive dystonia (DYT27) in addition to LGMD R22 (301). A mild phenotype of LGMD R22 caused by the COL6A3 mutation has been discovered that is secondary to aberrant assembly of vWF and for which MRI appears to be more diagnostic than muscle biopsy (471).

LGMD R23 laminin alpha-2-related – LAMA2. LAMA 2 encodes the extracellular matrix protein laminin alpha-2, a subunit of laminin 2 (merosin), the most abundant laminin isoform in the basement membrane of skeletal muscle cells. Laminin alpha-2 is expressed in the cardiac and skeletal muscles, central and peripheral nervous system, and skin.

Recessive mutations in LAMA2 cause a severe form of congenital muscular dystrophy (MDC1A). A limb-girdle muscular dystrophy presentation is associated with mutations causing reduced rather than absent levels of laminin alpha-2 on muscle biopsy. Patients present with mild mental retardation, white matter changes in brain MRI, dilated cardiomyopathy, and even peripheral neuropathy (83; 302; 118; 208), usually demyelinating. Migraines and epilepsy are other common presentations (313). Onset of symptoms in limb-girdle muscular dystrophy ranges from childhood to adulthood and some patients have independent ambulation and only mild symptoms (118).

Muscle MRI shows signal abnormalities in the adductor magnus and biceps femoris and concentric atrophy of muscles similar to that observed in collagen-VI related disorders, with outer area more affected than the center (in mild cases) (475; 83). The sartorius and gracilis muscles were spared. Besides muscle tissue, diagnosis can also be made with skin biopsy that will show absence or reduction of laminin alpha-2 at the dermal-epidermal junction. Brain MRI shows diffuse leukodystrophy but usually patients do not exhibit central nervous system symptoms. Symmetric high signal in the bilateral globus pallidus has also been described (208). Of note, patients with nonspecific brain MRI abnormalities and very subtle laminin alpha-2 staining have been described, making the diagnosis challenging in these cases (437).

Gene replacement using AAV9 vectors carrying the mini-agrin, which is an homologous functional substitute for mutated laminin alpha2 in dyw/dyw mice (LAMA2 muscular dystrophy model) partially amended nerve pathology as evidenced by improved motor function and sensorimotor processing, partial restoration of myelination, partial restoration of basement membrane via EM examination, as well as decreased regeneration of Schwann cells (412). Much research has been concentrated on MDC1A, but some results are likely applicable to LGMD R23 as well (177; 242; 260; 01; 205).

LGMD R24 POMGNT2-related – POMGNT2. O-linked mannose β-1,4-N-acetylglucosaminyltransferase 2 (POMGNT2) is also involved in glycosylation of alpha dystroglycan. Glycosylation of alpha dystroglycan is important for linking intracellular cytoskeleton to the extracellular matrix. POMGNT2 selectively catalyzes the first step toward the functional matriglycan structure of alpha dystroglycan based on the primary amino acid sequence in proximity to the site of O-mannosylation (203). Patients with POMGNT2 mutations have mild intellectual disability without brain malformation (133).

LGMD R25 blood vessel epicardial substance related – BVES. A homozygous nonsense variant of BVES in two Japanese patients caused loss of ambulation by midlife, cardiac involvement with symptomatic AV block, and severely affected posterior compartments of lower limbs and paraspinal muscles (235).

LGMD R26 Popeye domain-containing protein 3 related – POPDC3. This muscular dystrophy is related to dysfunction of Popeye domain-containing protein involved in modulating membrane trafficking of interacting proteins (564). These proteins are expressed in skeletal muscles and myocardium. Patients have proximal weakness in upper and lower extremities with mildly elevated CK. They can exhibit kyphosis deformity. Typical dystrophy in muscles may be absent. Calf hypertrophy is present (509). Myogenic changes are detected on EMG. MRI can show fat replacement in the gastrocnemius and paraspinals. Cardiac dysfunction is not usually seen.

LGMD R (number pending) - PYROXD1. This is a mild form of limb-girdle muscular dystrophy characterized by facial weakness, normal CK, and slow progression of weakness. The causative mutations include a homozygous missense variant c.464A>G, p.Asn155Ser, as well as a compound heterozygous frameshift variant c.329_332delTCTG, p.Leu112Valfs*8. It can be either child or adult onset. Cardiac involvement is present in some compound heterozygous cases (106).

Reclassified conditions (no longer considered in the current limb-girdle muscular dystrophy classification)

Myofibrillar myopathy (previously limb-girdle muscular dystrophy 1A) (5q) – myotilin. Myotilin is a thin-filament-associated protein located in the Z-band and binds alpha-actinin, gamma-filamin, and F-actin, and appears to help stabilize and anchor thin filaments during sarcomere assembly (434). Mutations in myotilin have also been documented in cases of myofibrillar myopathy, cardiomyopathy, peripheral neuropathy, and distal myopathy (455).

Mutations in myotilin were found in a West Virginian family of German descent with autosomal dominant limb-girdle muscular dystrophy previously linked to chromosome 5q. Affected members have a distinctive, nasal dysarthric speech pattern and involvement of both shoulder and pelvic muscles, starting in proximal lower more than upper extremities and progressing to distal muscles (213). A second family from Argentina with a myotilin mutation at Ser55Phe developed proximal limb weakness that spread to distal muscles with onset by age 42 to 58 years; 2 of 4 living patients had dysarthric speech, similar to the North American family (212). A novel myotilin mutation was found to underlie the defect in a large family with spheroid body myopathy (162). In a Spanish family, six patients were identified from three families who had clinical and pathologic findings that were intermediate between LGMD and myofibrillar myopathy (372). Myotilinopathy can also be unmasked by statin treatment (416).

Electromyography shows myopathic changes and abundant complex repetitive discharges in affected muscles. Muscle MRI shows hyperintensity in soleus and medial gastrocnemius. Muscles biopsy can show COX-negative fibers (416).

In a mouse model, myotilin mutations promote myofibrillar aggregation and subsequent contractile dysfunction (175). In this mouse model, overexpression of myotilin causes severe muscle degeneration, enhanced myofibrillar aggregation, and earlier onset of aggregation (174). This suggests that lowering total myotilin levels may be a therapeutic option in the future.

Emery-Dreifuss muscular dystrophy (EDMD) (previously limb-girdle muscular dystrophy 1B) (1q) – lamin A/C. Lamins are nuclear envelope proteins, as is emerin, the protein mutated in the more common X-linked form of Emery-Dreifuss muscular dystrophy. Lamin A is essential for anchoring emerin to the inner nuclear membrane and lamin C to the lamina, providing a possible link between the different forms of muscular dystrophies with cardiac involvement (518). Thus, defective assembly of the nuclear lamina is one common feature of all these diseases. The “structural” hypothesis suggests that mutated A-type lamins or the associated nuclear envelope proteins disrupt the integrity of the cell nuclear membrane, resulting in nuclear breakage and cell death in tissues exposed to mechanical stress, such as muscle fibers. The “gene regulation hypothesis” suggests that A-type lamins are crucial in tissue-specific gene expression. Indeed, in Emery-Dreifuss muscular dystrophy muscle, the transcriptional regulation is defective, likely due to a focal loss and disorganization of heterochromatin in fibroblast and muscle fiber nuclei (310). The amount of lamin A/C appears to correlate with phenotypic severity. Ho and colleagues found that both lamin A/C and emerin regulate a downstream protein mechanosensitive transcription factor megakaryoblastic leukemia 1 (MKL1), a myocardin family member that is pivotal in cardiac development and function, based on mouse models and mutant cell lines (223). Lamin A/C and emerin may regulate gene expression through modulation of nuclear and cytoskeletal actin polymerization.

Lamins A and C are alternate splice forms from the same LMNA gene. These proteins have a role in several cellular processes, and mutations in LMNA are associated with a wide range of disease phenotypes, from neuromuscular, cardiac, and metabolic disorders to premature aging syndromes; however, no clear genotype-phenotype correlation is described in the current literature. Even within patients with muscle involvement, different phenotypes have been reported: limb-girdle muscular dystrophy (formerly LGMD1B), autosomal dominant Emery-Dreifuss muscular dystrophy 2, and a form of congenital muscular dystrophy.

Onset of weakness is usually in the 3rd or 4th decade (but as early as 1st decade) with predominant weakness of pelvic muscles. Facial weakness and scapular winging can be present. In a large Italian case series, contractures, traditionally associated with Emery-Dreifuss muscular dystrophy 2, were observed in 24 of 37 patients. In 13 patients, contractures were localized at multiple sites and in 10 developed before obvious clinical signs of muscle weakness (311). Hypermobility of the interphalangeal and metacarpophalangeal joints can also be present (539). The primary differentiating feature from other forms of limb-girdle muscular dystrophy is cardiac involvement, including conduction disturbances, arrhythmias, and syncope (513) with risk of sudden cardiac death, requiring pacemaker, defibrillator placement, or heart transplantation in some cases. Embolic ischemic stroke secondary to cardiac arrhythmia can be the presentation (87). A case of late onset (65 years of age) with paraspinal muscle pseudohypertrophy has also been reported (170).

Some LMNA mutation patients present initially with dilated cardiomyopathy or familial cardiac conduction disease (514). A particularly malignant mutation of the LMNA gene (c.908-909delCT) can cause rapid atrioventricular conduction abnormalities and sudden death (08). Other LMNA mutation patients can have an atypical phenotype characterized by isolated humeral, peroneal, or quadriceps weakness, in some cases with contractures or head drop syndrome (311).

Similar to other forms, there is phenotypic variability. In a study of four patients with mutations in the same LMNA exon 11 codon, one patient had congenital onset; another had severe, early cardiac involvement; a third patient had cardiac involvement that long predated skeletal muscle weakness; and the last patient had mild, late-onset limb-girdle muscular dystrophy presentation (329).

Muscle MRI shows that the adductor magnus, semimembranosus, and short and long head of the biceps femoris are the most frequently involved muscles in laminopathies whereas the gracilis and sartorius are less involved. The vasti muscles are usually involved, with sparing of the rectus femoris (296).

Muscle biopsy can show myocardial fibrosis or unspecific inflammatory signs in microscopic examination of heart or skeletal muscle in some patients (309). Fiber-type disproportion (430) and myopathic, dystrophic and myofibrillar changes with desmin accumulation have also been described (311).

Lamin defects are also implicated in a form of inherited dilated cardiomyopathy with conduction defect (CMD1A), familial partial lipodystrophy (151), mandibuloacral dysplasia, Hutchinson Gilford progeria (110), and a form of hereditary axonal motor and sensory neuropathy (CMT type 2) (111; 85). Overlapping phenotypes have been reported. Vital and colleagues described a patient with a phenotypic and pathologic combination of axonal polyneuropathy and myopathy caused by a dominant missense lamin A/C mutation, E33D (530). Another report described a young girl with progeria, congenital myopathy, and cardiac involvement due to a de novo missense mutation in the LMNA gene (S143F) (265).

Rippling muscle disease (previously limb-girdle muscular dystrophy 1C) (3p25) – caveolin 3. Mutations in the CAV3 gene were found in two families with dominant limb-girdle muscular dystrophy linked to chromosome 3p25 (338). Caveolins are major structural components of caveolae that serve as signaling hubs and membrane reservoirs at the cell surface. They also function as scaffolding proteins that organize and enrich caveolin-interacting proteins and lipids. During muscle contraction, caveolae are passively flattened and compensate plasma membrane stretch by extending the cell surface. Caveolin-3, a 21- to 24-kDa protein mainly expressed in muscle fibers, is the principal structural protein of caveolar membrane domains in skeletal, smooth, and heart muscle; it localizes at the sarcolemma within caveolae, associates with the dystroglycan complex, and binds to dysferlin.

Symptoms often begins around 5 years of age with cramping, mild weakness, and calf hypertrophy, but mild cases with clinical weakness are also seen (72). Percussion-induced muscle mounding can also be seen (386).

Caveolin 3 mutations also produce other phenotypes besides limb-girdle muscular dystrophy. The CAV3 gene has been implicated in nondystrophic rippling muscle disease, idiopathic and familial isolated hyperCKemia, a form of distal myopathy, long QT syndrome, and hypertrophic cardiomyopathy (72; 42; 332; 494; 550; 452). An autosomal recessive phenotype has also been reported in a 57-year-old woman with a novel homozygous intronic mutation in the CAV3 gene (352). Myalgia, exertion intolerance, and recurrent rhabdomyolysis are features associated with CAV3 mutations. Rippling muscle contraction and percussion-induced rapid muscle contractions (PIRCs) are important clinical clues indicative of caveolinopathies (446). In patients with CAV3-related distal myopathy, atrophy can be symmetric or asymmetric, usually in lower extremities (although cases with hand involvement have been described) (89).

Muscle MRI of patients with rippling muscle disease can be normal or show preferential and early involvement of rectus femoris (sometimes with peripheral hyperintensity), semitendinosus, and gastrocnemius (236). EMG can show electrical myotonic discharges (337).

One screen of 663 patients with a variety of neuromuscular phenotypes found CAV3 mutations in roughly 1% (169). The same group found that, in contrast to diminished levels in most fibers, CAV3 overexpression in regenerating fibers appears to lead to multiple misfolded oligomers in the Golgi complex but improved membrane immunostaining. Caveolin-3 appears to interact with dysferlin; accordingly, in some patients with dysferlinopathy there is a secondary reduction in the levels of caveolin-3 (534). A toxic gain of function exerting stress on the endoplasmic reticulum may underlie the disease mechanism based on a transgenic mouse model (276).

Myofibrillar myopathy (previously limb-girdle muscular dystrophy 1E) (2q) – desmin. This subtype is associated with cardiac failure and conduction system disease and previously was descriptively named "familial dilated cardiomyopathy with conduction disease and myopathy." Later onset patients generally present before 25 years of age and remain ambulatory (334). Creatine kinase is typically elevated. The genetic defect is determined to be due to an intron splice donor site mutation of the desmin gene located on chromosome 2q35 (189). Desminopathies usually manifest in the second to the fourth decade of life. The main features are lower and later upper limb muscle weakness slowly spreading to involve truncal, neck flexor, facial, bulbar, and respiratory muscles. The patients may have arrhythmias and restrictive, dilated, or hypertrophic cardiomyopathy. Desmin myopathies are a diverse group of disorders representing a subcategory of myofibrillar myopathies and some other entities such as Kaeser scapuloperoneal syndrome, myofibrillar myopathy with arrhythmogenic right ventricular cardiomyopathy (ARVD7), myofibrillar myopathy (MFM1), and dilated cardiomyopathy (1F and 1I). A recessive form of limb girdle muscular dystrophy without cardiomyopathy or myofibrillar myopathy has also been described (81).

Unconfirmed (previously limb-girdle muscular dystrophy 1H) (3p23-25) – Unknown protein product. A novel form of autosomal dominant limb-girdle muscular dystrophy associated with rimmed vacuoles is proposed to be type 1H. Bisceglia and colleagues investigated a novel Italian family with 11 of 19 affected members (45). Five subjects presented with slowly progressive proximal arm and leg weakness beginning during the fourth to fifth decade. Earlier disease onset was observed in a group of patients presenting with muscle weakness or calf hypertrophy, or occasionally high creatine kinase and lactate serum levels. Two muscle biopsies showed morphological findings compatible with muscular dystrophy associated with subsarcolemmal accumulation of mitochondria and the presence of multiple mitochondrial DNA deletions. Several candidate genes in the region identified were excluded (45).

Myofibrillar myopathy (previously limb-girdle muscular dystrophy 2R) (2q) – Desmin. Desmin is a type III intermediate filament found near the Z disk in sarcomeres. Desminopathies usually manifest in the second to the fourth decade of life with lower and later upper limb muscle weakness slowly spreading to involve truncal, neck flexor, facial, bulbar, and respiratory muscles. The patients may have arrhythmias and restrictive, dilated, or hypertrophic cardiomyopathy. Desminopathies are inherited with an autosomal dominant pattern or result from de novo mutations; autosomal recessive mutations are rare. In muscle biopsy, there are findings of myofibrillar myopathy with presence of pleomorphic amorphous abnormal fiber areas containing aggregates, including desmin and other proteins such as dystrophin, plectin, alpha B-crystallin, gelsolin, actin, and ubiquitin. In addition, an autosomal dominant form also exists (see above). In a Turkish family with two affected siblings, onset of weakness varied from 15 to 27 years of age, with progressive severe proximal more than distal weakness and mild facial weakness. One became wheelchair-bound at age 33 years. There was scapular winging but no respiratory involvement or scoliosis. No cardiac symptoms were reported, but one patient had abnormal EKG. CK was normal. Muscle biopsy showed dystrophic changes but no changes of myofibrillar myopathy, different from other desminopathy phenotypes. The c.1289-2A>G mutation identified in these two patients is postulated to interfere with desmin-lamin B interactions (81).

Pompe disease (previously limb-girdle muscular dystrophy 2V) (17q) – GAA. GAA encodes the lysosomal enzyme acid alpha glucosidase, and mutations are the cause of Pompe disease. Late-onset Pompe disease can present as limb-girdle muscular dystrophy. The red flags for diagnosis of Pompe disease in patients with limb-girdle muscular dystrophy are: (1) mild, nondystrophic, myopathic features on muscle biopsy, often missing the typical vacuoles and glycogen accumulation; (2) CK levels below 1000; and (3) disproportionate axial and respiratory muscle involvement in comparison with limb muscle involvement. Myotonic discharges in paraspinal muscles are also suggestive of this diagnosis. Some patients present with idiopathic hyperCKemia (410; 196).

LIMS2-related myopathy (2q) – LIMS2. LIMS is an evolutionarily conserved protein, critical for muscle attachment. LIMS, along with integrin linked kinase (ILK) and parvin, is part of a ternary complex known as ILK–Pinch–Parvin (IPP), a core component of the integrin-actin cytoskeleton, and functions as a signaling mediator that transmits mechanical signals to downstream effectors. Two siblings with limb-girdle muscular dystrophy have been described. Weakness started at 5 years of age and progressed to wheelchair dependence by age 12 years. Dilated cardiomyopathy and macroglossia less severe at the tip with an appearance of a triangular tongue was also noted. CK was elevated. On cardiac MRI, both siblings also demonstrated a subepicardial rim of delayed enhancement predominantly in the inferolateral wall, likely representing fibrosis (86).

POPDC1-related myopathy (6q) – POPDC1. POPDC1, which is also known as BVES, is a member of the Popeye domain-containing (Popdc) gene family, encoding transmembrane proteins, which is highly expressed in cardiac and skeletal muscle in an overlapping manner. POPDC proteins possess the evolutionarily conserved Popeye domain, which functions as a high-affinity cAMP-binding site. POPDC proteins are localized primarily at the plasma membrane and in T-tubules, although they have also been found at the nuclear envelope of striated muscle cells. Mutation in this gene in four family members with limb-girdle muscular dystrophy and cardiac arrhythmia, including AV block, bradycardia-causing syncope, has been described. CK was elevated. Onset of weakness in one patient was in the fifth decade (448).

TOR1AIP1-related myopathy (previously limb-girdle muscular dystrophy 2Y) (1q) – TOR1AIP1. TOR1AIP1 encodes lamina-associated polypeptide-1 (LAP1B) and is a type II integral protein localized to the inner nuclear membrane through a single transmembrane domain. LAP1B has been shown to interact with the nuclear envelope protein emerin, and loss of LAP1B is associated with dystrophic changes on muscle biopsy and early lethality in knockout mice.

Reported cases present with proximal and distal weakness and atrophy, rigid spine, and contractures of the proximal and distal interphalangeal hand joints. Onset of weakness is in the first or second decades. Additionally, cardiomyopathy and respiratory involvement were noted. CK is mildly elevated. Cardiac failure can be severe, requiring heart transplant (257; 179).

Classification of limb-girdle muscular dystrophies. A reclassification of limb-girdle muscular dystrophy was proposed by the European Neuromuscular Center (ENMC) LGMD workshop study group. This new classification follows the formula: “LGMD, inheritance (R or D), order of discovery (number), affected protein” (477). Because of the definition of limb-girdle muscular dystrophy, that needs to have been described in at least two different families, some conditions are no longer considered limb-girdle muscular dystrophy (only one family described in the literature). Table 1 includes the new and previous classification for clarity purposes. Distinguishing clinical features (if they exist) are also included in the table.

Table 1. Classification of Limb-Girdle Muscular Dystrophy

New nomenclature	Old nomenclature	Gene	Distinguishing clinical features	Reason for exclusion in new classification
LGMD D1 DNAJB6-related	LGMD 1D	7q36 DNA JB6	Foot drop, rimmed vacuoles, myofibrillar aggregates, myotonia in EMG (some cases)
LGMD D2 TNP03-related	LGMD 1F	7q32.1-q32.2 TNPO3--	Early contractures, arachnodactyly, macroglossia, dysarthria, pes cavus, calf hypertrophy, gynecomastia, worsening of weakness (after intense exercise, alcohol use, pregnancy), ophthalmoparesis, rimmed vacuoles, acid phosphatase vacuoles, cytoplasmic inclusions, ragged red fibers, mitochondrial paracrystaline inclusions
LGMD D3 HNRNPDL-related	LGMD 1G	4q21 HNRNPDL--	Limited finger and toe flexion, cataracts, scapular winging, rimmed vacuoles, autophagic vacuoles
LGMD D4 calpain3-related	LGMD 1I	15q CAPN3	Calpainopathy; scapular winging, abdominal hernia, severe hip adductor weakness, toe walking, ankle contractures, absence of calf hypertrophy, myalgia, British, Southern or Eastern European, or Brazilian ancestry, asymmetry
LGMD D5 collagen 6-related	Bethlem myopathy dominant	21q, 2q COL6A1, COL6A2, COL6A3	Early onset with contractures, distal joint hyperlaxity, keratosis pilari, keloids
LGMD R1 calpain3-related	LGMD 2A	15q CAPN3	Calpainopathy; abdominal hernia, severe hip adductor weakness, toe walking, ankle contractures, scapular winging, absence of calf hypertrophy, British, Southern or Eastern European, or Brazilian ancestry, asymmetry
LGMD R2 dysferlin-related	LGMD 2B	2q DYSF	Dysferlinopathy biceps lump and diamond on quadriceps, high CK, dysferlin—calf atrophy and asymmetry
LGMD R3 α-sarcoglycan-related	LGMD 2D	17q SGCA	Alpha-sarcoglycan/adhalin--variable severity, cardiac respiratory involvement, hip adductor weakness, and muscle hypertrophy, high CK
LGMD R4 β-sarcoglycan-related	LGMD 2E	4q SGCB	Beta-sarcoglycan--cardiac, respiratory involvement, hip adductor weakness, muscle hypertrophy, macroglossia, high CK
LGMD R5 γ -sarcoglycan-related	LGMD 2C	13q SGCG	Gamma-sarcoglycan severe childhood limb-girdle muscular dystrophy--macroglossia, ankle contractures, scoliosis, cardiac, respiratory involvement, hip adductor weakness, muscle hypertrophy, high CK
LGMD R6 δ-sarcoglycan-related	LGMD 2F	5q SGCD	Delta-sarcoglycan--cardiac respiratory involvement, hip adductor weakness, muscle hypertrophy high CK
LGMD R7 telethonin-related	LGMD 2G	17q TCAP	Telethoninopathy—calf hypertrophy, cardiac involvement, rimmed vacuoles
LGMD R8 TRIM 32-related	LGMD 2H	9q TRIM32	Hutterites, scapular winging
LGMD R9 FKRP-related	LGMD 2I	19q FKRP	Northern European ancestry, scapular winging, calf hypertrophy, early cardiorespiratory involvement, and myoglobinuria
LGMD R10 titin-related	LGMD 2J	2q TTN	Finnish and French populations, rimmed vacuoles
LGMD R11 POMT1-related	LGMD 2K	9q34.1 POMT1
LGMD R12 anoctamin5-related	LGMD 2L	11p ANO5	Calf atrophy and myoglobinuria
LGMD R13 Fukutin-related	LGMD 2M	9q FKTN	CNS involvement, learning difficulties, and muscle hypertrophy
LGMD R14 POMT2-related	LGMD 2N	1p POMT2	CNS involvement, learning difficulties, and muscle hypertrophy
LGMD R15 POMGnT1-related	LGMD 2O	14q POMGnT1	CNS involvement, learning difficulties, and muscle hypertrophy
LGMD R16 α-dystroglycan-related	LGMD 2P	3p DAG1
LGMD R17 plectin-related	LGMD 2Q	8q PLEC	Epidermolysis bullosa
LGMD R18 TRAPPC11-related	LGMD 2S	4q TRAPPC11	CNS involvement, movement disorders, alacrima, achalasia, cataracts, and hepatic steatosis
LGMD R19 GMPPB-related	LGMD 2T	3p GMPPB	CNS involvement, neuromuscular transmission defects, cataracts rhabdomyolysis
LGMD R20 ISPD-related	LGMD 2U	7p ISPD	CNS involvement, calf hypertrophy, and scapular winging
LGMD R21 POGLUT1-related	LGMD 2Z	3q POGLUT1
LGMD R22 collagen 6-related	Bethlem myopathy recessive	21q, 2q COL6A1, COL6A2, COL6A3	Early onset with contractures
LGMD R23 laminin α2-related	Laminin α2-related muscular dystrophy	LAMA2
LGMD R24 POMGNT2-related	POMGNT2-related muscular dystrophy	POMGnT2
LGMD R25 BVES-related	Blood vessel epicardial substance	BVES	Loss of ambulation, AV block, posterior compartment of lower limbs and paraspinals
LGMD R26 Popeye domain-containing protein 3 related	Popeye domain-containing protein 3 related	POPDC3	Proximal weakness, fatty replacement in the gastrocnemius and paraspinals
Reclassified conditions (no longer included in the new LGMD classification)
Myofibrillar myopathy	LGMD 1A	5q MYOT	Nasal dysarthria, hypophonia, asymmetric muscle weakness and atrophy rimmed vacuoles, occasional nemaline rod-like inclusions	Distal weakness
Emery–Dreifuss muscular dystrophy (EDMD)	LGMD 1B	1q LMNA	Contractures, cardiac involvement, cardiac conduction abnormalities, and humeroperoneal weakness	High risk of cardiac arrhythmias; EDMD phenotype
Rippling muscle disease	LGMD 1C	3p CAV3	Rippling muscles, percussion-induced rapid contractions, prominent muscle cramps, and calf hypertrophy	Main clinical features rippling muscle disease and myalgia
Myofibrillar myopathy	LGMD 1E	6q DES	Foot drop, cardiac conduction abnormalities	Primarily false linkage; distal weakness and cardiomyopathy
?	LGMD 1H	Not confirmed		False linkage
Myofibrillar myopathy	LGMD 2R	6q DES	Foot drop, cardiac conduction abnormalities	Distal weakness
Pompe disease	LGMD 2V	17q GAA	Axial and respiratory muscle involvement	Known disease entity, histological changes
PINCH-2 related myopathy	LGMD 2W	PINCH2		Reported in one family
BVES related myopathy	LGMD 2X	BVES		Reported in one family
TOR1AIP1 related myopathy	LGMD 2Y	LGMD 2Y		Reported in one family

Table 2. Allelic Disorders with Limb-Girdle Muscular Dystrophy Gene Product Mutations

New nomenclature	Old nomenclature	Gene	Allelic disorders
LGMD D1 DNAJB6-related	LGMD 1D	7q36 DNA JB6	Distal dystrophy with rimmed vacuoles and cardiomyopathy susceptibility
LGMD D2 TNP03-related	LGMD 1F	7q32.1-q32.2 TNPO3--
LGMD D3 HNRNPDL-related	LGMD 1G	4q21 HNRPDL--
LGMD D4 calpain3-related	LGMD 1I	15q CAPN3	HyperCKemia
LGMD D5 collagen 6-related	Bethlem myopathy dominant	21q, 2q COL6A1, COL6A2, COL6A3	Ullrich congenital muscular dystrophy
LGMD R1 calpain3-related	LGMD 2A	15q CAPN3	HyperCKemia
LGMD R2 dysferlin-related	LGMD 2B	2q DYSF	Miyoshi myopathy HyperCKemia Anterior compartment myopathy Distal myopathy with anterior tibialis onset (DMAT)
LGMD R3 α-sarcoglycan-related	LGMD 2D	17q SGCA	Exercise intolerance
LGMD R4 β-sarcoglycan-related	LGMD 2E	4q SGCB
LGMD R5 γ -sarcoglycan-related	LGMD 2C	13q SGCG
LGMD R6 δ-sarcoglycan-related	LGMD 2F	5q SGCD	Dilated cardiomyopathy 1L
LGMD R7 telethonin-related	LGMD 2G	17q TCAP	Dilated cardiomyopathy 1N Congenital muscular dystrophy Hypertrophic cardiomyopathy
LGMD R8 TRIM 32-related	LGMD 2H	9q TRIM32	Sarcotubular myopathy Bardet-Biedl syndrome type 11
LGMD R9 FKRP-related	LGMD 2I	19q FKRP	Congenital muscular dystrophy with muscle hypertrophy and normal CNS Myopathy with abnormal merosin Walker-Warburg syndrome
LGMD R10 titin-related	LGMD 2J	2q TTN	Dominant: Distal myopathy (Finnish) Myopathy with early respiratory failure Cytoplasmic aggregate myopathy Dilated cardiomyopathy 1G Recessive: Congenital myopathy (multicore disease, congenital myopathy with fatal dilated cardiomyopathy) Centronuclear myopathy Fiber type size disproportion Emery-Dreiffus without cardiac involvement Hypertrophic cardiomyopathy
LGMD R11 POMT1-related	LGMD 2K	9q34.1 POMT1	Walker-Warburg syndrome
LGMD R12 anoctamin5-related	LGMD 2L	11p ANO5	Nondysferlin Miyoshi myopathy Gnathodiaphyseal dysplasia Normal strength with high CK
LGMD R13 Fukutin-related	LGMD 2M	9q FKTN	Fukuyama muscular dystrophy Cardiomyopathy Muscle eye brain disease Congenital muscular dystrophy
LGMD R14 POMT2-related	LGMD 2N	1p POMT2	Walker-Warburg syndrome Congenital muscular dystrophy with mental retardation
LGMD R15 POMGnT1-related	LGMD 2O	14q POMGnT1	Muscle eye brain disease Congenital muscular dystrophy
LGMD R16 α-dystroglycan-related	LGMD 2P	3p DAG1	Asymptomatic high CK Congenital muscular dystrophy
LGMD R17 plectin-related	LGMD 2Q	8q PLEC	Congenital muscular dystrophy with familial junctional epidermolysis bullosa Muscular dystrophy and myasthenic syndrome
LGMD R18 TRAPPC11-related	LGMD 2S	4q TRAPPC11
LGMD R19 GMPPB-related	LGMD 2T	3p GMPPB	Walker-Warburg syndrome Congenital muscular dystrophy Congenital myasthenic syndrome
LGMD R20 ISPD-related	LGMD 2U	7p ISPD	Congenital muscular dystrophy Limb-girdle muscular dystrophy with cerebellar involvement
LGMD R21 POGLUT1-related	LGMD 2Z	3q POGLUT1
LGMD R22 collagen 6-related	Bethlem myopathy recessive	21q, 2q COL6A1, COL6A2, COL6A3	Ullrich congenital muscular dystrophy
LGMD R23 laminin α2-related	Laminin α2-related muscular dystrophy	LAMA2
LGMD R24 POMGNT2-related	POMGNT2-related muscular dystrophy	POMGnT2
Reclassified conditions (no longer included in the new LGMD classification)
Myofibrillar myopathy	LGMD 1A	5q MYOT	Myofibrillary myopathy Distal myopathy Cardiomyopathy Peripheral neuropathy Spheroid body myopathy
Emery–Dreifuss muscular dystrophy (EDMD)	LGMD 1B	1q LMNA	Autosomal dominant Emery-Dreifuss muscular dystrophy Cardiomyopathy (CMD1A) Familial partial lipodystrophy Mandibuloacral dysplasia Hutchinson Gilford progeria Hereditary axonal motor and sensory neuropathy (CMT type 2) Quadriceps myopathy with dilated cardiomyopathy Congenital muscular dystrophy with rigid spine
Rippling muscle disease	LGMD 1C	3p CAV3	Rippling muscle disease Distal myopathy HyperCkemia Familial hypertrophic cardiomyopathy Long QT syndrome Proximal weakness or exertional myalgias with calf hypertrophy Muscle pain/exercise intolerance and rhabdomyolysis
Myofibrillar myopathy	LGMD 1E	6q DES	Dominant myopathies (Kaeser scapuloperoneal syndrome Myofibrillar myopathy Distal myopathy, cardiomyopathy, and autophagic vacuoles) Dominant cardiomyopathies (dilated cardiomyopathy 1F and 1I, ARVD7) Recessive myopathies (LGMD2R, myopathy early and adult onset)
?	LGMD 1H	Not confirmed
Myofibrillar myopathy	LGMD 2R	6q DES	Dominant myopathies (Kaeser scapuloperoneal syndrome, myofibrillar myopathy, distal myopathy, cardiomyopathy, and autophagic vacuoles); Dominant cardiomyopathies (dilated cardiomyopathy 1F and 1I, ARVD7) Recessive myopathies (LGMD2R, myopathy early and adult onset)
Pompe disease	LGMD 2V	17q GAA
PINCH-2 related myopathy	LGMD 2W	PINCH2
BVES related myopathy	LGMD 2X	BVES
TOR1AIP1 related myopathy	LGMD 2Y	1q TOR1AIP1

Mechanisms of disease brought about by the alterations in the numerous genes thus far identified are complex and vary between subtypes. Muscular dystrophy can result from diverse defects in proteins of different muscle cell compartments including extracellular matrix (collagen VI, merosin), cell membrane (dystrophin, sarcoglycans, caveolin-3, dysferlin, integrins), nuclear envelope (lamins, emerin), and with different functions, such as cellular enzymes (calpain-3), and organelle or sarcomere structural integrity (telethonin and others). Ongoing work is aimed at determining how previously known and recently uncovered gene defects and proteins affect myofiber physiology and ultimately lead to muscle cell death. Also awaiting explanation is why cell death occurs at a particular age, in predisposed muscles, and to variable degrees in different subtypes and individuals. There is evidence that the final pathways include apoptosis and muscle necrosis, but the processes that lead to this end remain to be determined. Other general processes that can be disrupted include structural integrity of the membrane, other signaling properties, cellular trafficking, myofiber assembly, nuclear envelope function, connective tissue structure, and likely a number of others.

Epidemiology

Most limb-girdle muscular dystrophies are inherited as autosomal recessive traits; autosomal dominant limb-girdle muscular dystrophy is relatively uncommon.

Limb-girdle muscle dystrophy is considered the fourth most common muscular dystrophy with a pooled prevalence of 1.63 per 100,000 (range 0.56-5.75 per 100,000) after dystrophinopathies, myotonic dystrophies, and fascioscapulohumeral muscular dystrophy (314).

Autosomal dominant limb-girdle muscular dystrophy is relatively rare and has been reported primarily in isolated families (362; 473; 40; 11; 92; 343; 486; 382). Although the exact incidence of autosomal dominant limb-girdle muscular dystrophy is unknown, no more than 5% to 10% of all limb-girdle dystrophies are presumed to be dominant (64).

In the United States, a study using next-generation sequencing in 4656 patients established diagnosis in 27% of cases (1259 patients). The major contributing genes to limb-girdle muscular dystrophy phenotypes were CAPN3 (17%), DYSF (16%), FKRP (9%), and ANO5 (7%) (360). Calpainopathy (LGMD R1, CAPN3 mutation) is the most common limb-girdle muscular dystrophy in American and European countries except Denmark, where limb-girdle muscular dystrophy FKRP mutations are the most common. An epidemiological study found that Norway has a relatively high rate of limb-girdle muscular dystrophy in comparison to the rest of Europe, at a rate of 12.8 cases per 100,000 people, with CAPN3, ANO5, and FKRP mutations as the most common (353).

Prevention

All limb-girdle dystrophies are inherited diseases involving skeletal muscle, and in some cases additional organ systems. Typically, no risk factor exists in the environment; however, the mechanisms that determine the severity and expression of many of the disorders have yet to be determined, such as whether secondary genetic factors or as yet other unknown influences play a role. To prevent the birth of an affected child in families with known defective genes, appropriate genetic counseling is advocated if possible. Judging from the current pace of research, specific and reliable diagnostic tests for limb-girdle muscular dystrophy should be available in the near future for effective prenatal disease detection. In families with autosomal recessive limb-girdle muscular dystrophy, consanguineous marriages must obviously be discouraged.

Differential diagnosis

Duchenne dystrophy and Becker dystrophy and female manifesting heterozygotes for Duchenne dystrophy can be indistinguishable from autosomal recessive limb-girdle muscular dystrophy and must be excluded, especially in sporadic limb-girdle muscular dystrophy cases. In fact, the diagnosis of sporadic limb-girdle muscular dystrophy should not be made unless dystrophinopathy has been excluded by appropriate laboratory tests (51; 521; 299). DNA analysis for dystrophin gene mutations and staining for dystrophin in muscle biopsy specimens are both important means to identify the various dystrophinopathies, including manifesting carriers. This condition is especially relevant in girls with a limb-girdle muscular dystrophy pattern and no manifesting male dystrophinopathy relatives. Golla and colleagues identified five such cases (183). The electrocardiogram, if abnormal, may also help in identifying female carriers of Duchenne dystrophy (131; 55), although it can be abnormal in some limb-girdle muscular dystrophy phenotypes.

X-linked Emery-Dreifuss muscular dystrophy differs from limb-girdle muscular dystrophy in inheritance (X-linked), characteristic humeroperoneal distribution of weakness, early contractures, and cardiac abnormalities (132). This dystrophy is caused by alterations in the emerin gene mapped to the chromosome Xq-locus (44; 267). However, the rarer dominant form, secondary to mutations in the lamin A/C gene, may be confused with some dominant forms of limb-girdle muscular dystrophy (52; 350).

Facioscapulohumeral dystrophy is inherited as an autosomal dominant trait, and, unlike limb-girdle muscular dystrophy, facial muscles are affected early in the course of the disease. Asymmetric scapular weakness associated with distal lower limb weakness, pronounced lower abdominal weakness, and lumbar hyperlordosis are suggestive of facioscapulohumeral muscular dystrophy in the differential diagnosis with limb-girdle muscular dystrophy. Diagnosis by genetic analysis is now widely available. Scapuloperoneal syndrome is a heterogeneous disorder, but the distinctive distribution of weakness distinguishes it from limb-girdle muscular dystrophy (258). In familial neurogenic scapuloperoneal syndrome, EMG and muscle biopsy typically show a neurogenic process (490). Myotonic dystrophy is usually evident on the basis of the characteristic topography of muscle weakness, myotonia, and multisystem involvement although myotonic dystrophy type 2 can have subtle clinical findings and absent clinical or electrical myotonia in some cases.

Many congenital myopathies, including centronuclear myopathy (464; 56), nemaline rod myopathy (463), central core disease (123), multicore disease (134), congenital fiber-type disproportion (58), desmin myopathy (225), and myopathy with tubular aggregates (426) may present with proximal muscle weakness in childhood or adult life. The weakness in congenital myopathies may be gradually progressive or relatively static and may be confused with some forms of limb-girdle muscular dystrophy. A core-rod myopathy mimicking limb-girdle muscular dystrophy secondary mutations in KBTBD13 starting at age 50 has also been described (173). Careful histochemical studies of muscle specimen will clarify the diagnosis of congenital myopathies and usually identify the form.

Patients with congenital myasthenic syndromes can also be misdiagnosed with limb girdle muscular dystrophy or congenital muscular dystrophies. This distinction is important as congenital myasthenic syndromes are often treatable (249). Electrophysiological examination with repetitive nerve stimulation is valuable in confirming this diagnosis. Genetic testing is available for most of the subtypes.

The glycogenoses and the lipid myopathies may also present with proximal muscle weakness in different age groups, but these are usually differentiated from limb-girdle muscular dystrophy by histological and biochemical studies of muscle (115).

The mitochondrial myopathies represent a large and heterogeneous group of disorders that may have various morphological and biochemical characteristics (115). Although some mimic limb-girdle muscular dystrophy, most mitochondrial syndromes are multisystemic, and specific molecular tests are available for many forms.

In some cases, it may be difficult to separate spinal muscular atrophies from muscular dystrophies. In particular, Kugelberg-Welander spinal muscular atrophy can closely mimic limb-girdle dystrophy (277). The presence of fasciculations, relatively diffuse weakness, normal serum enzymes, and classic neurogenic findings on EMG and muscle biopsy differentiate the spinal muscular atrophies from muscular dystrophies.

Limb-girdle muscular dystrophy may be misdiagnosed as idiopathic inflammatory myopathy as the symptoms and MRI findings overlap among them (378). Although the fatty replacement in limb-girdle muscular dystrophy and muscular edema are expected to be present in idiopathic inflammatory myopathy as characteristic findings, it has been noted that fatty replacement and edema can coexist in limb-girdle muscular dystrophy and idiopathic inflammatory myopathy. A study by Hsu and colleagues showed that fatty replacement is more prevalent in limb-girdle muscular dystrophy (226). MRI, particularly of the adductor magnus, in all patients with limb-girdle muscular dystrophy showed fatty replacement with no edema (226). Larger studies are needed to confirm that the adductor magnus muscle could provide a biosignature to categorizing limb-girdle muscular dystrophy. Another study highlighted that abundant p62 staining and muscle edema affecting the adductor magnus on MRI seem to be good markers for immune-mediated necrotizing myopathy (555). Once a limb-girdle muscular dystrophy syndrome is suspected, a combination of muscle biopsy analysis, analysis of protein components of muscle (Western blot), and molecular genetics is usually needed for definitive confirmation of a particular subtype. In rare cases a limb-girdle muscular dystrophy syndrome is highly suspected in an individual family, but no linkage to any known site is seen. Such new pedigrees are highly sought in order to identify and further characterize new genes and disease mechanisms.

Confusing conditions

Proximal muscle weakness is the common clinical hallmark of most hereditary and acquired myopathies, and many can mimic limb-girdle muscular dystrophy. Laboratory findings in limb-girdle muscular dystrophy are not specific and resemble those of other muscular dystrophies. The large number of apparently isolated cases may obscure autosomal recessive pattern of inheritance.

Therapeutically, important disorders to differentiate from limb-girdle muscular dystrophy include polymyositis and dermatomyositis. In most cases, specific clinical and laboratory features identify an inflammatory muscle disease. Relatively rapid evolution, more generalized weakness (often involving muscles of the neck and pharynx), abundant spontaneous activity on EMG, inflammatory changes on muscle biopsy, and response to immunosuppressive therapy are all important distinguishing features. Exceptions occur, however, because some cases of polymyositis lack a few or many characteristic clinical and laboratory features and can simulate limb-girdle muscular dystrophy. A high index of suspicion, close clinical monitoring, and adequate clinical trial of corticosteroid therapy in suspected cases of polymyositis are important discriminators. The presence of myositis specific antibodies, such as Jo-1, SRP, MDA5, etc., is highly specific for myositis and not for genetic disease (317).

Myopathy associated with anti-HMGCR (3-hydroxy-3-methylglutarylcoenzyme A reductase) antibodies can mimic limb-girdle muscular dystrophy or present with asymptomatic hyperCKemia. Testing for anti-HMGCR antibodies should be considered in patients with limb-girdle muscular dystrophy or asymptomatic hyperCKemia phenotype, and negative genetic testing as immunotherapy is associated with clinical improvement (342).

Dysferlinopathy is probably most likely to simulate a steroid-resistant inflammatory myopathy, in part because of the high CK levels and inflammatory changes in muscle biopsy. Another treatable condition that can present with limb-girdle weakness is acid maltase deficiency (adult onset Pompe disease). This can be confirmed with measurement of the alpha-glucosidase activity in dry blood spot and with genetic testing. LGMD R1 and R2 are easily misdiagnosed as inflammatory myopathies as they are very similar in clinical manifestation and muscle pathology (378; 541).

Inclusion-body myositis is an inflammatory myopathy that may be confused with limb-girdle muscular dystrophy. Although the disease typically presents later in life, childhood-onset cases have been described (423). Familial inclusion-body myopathy is generally a more distal quadriceps-sparing process (481), but proximal weakness in limb-girdle distribution can occur; the gene for this disorder, a sialic acid synthesis enzyme, is now known (324; 128). Similar to muscular dystrophy, but unlike polymyositis or dermatomyositis, hypertrophied muscle fibers can be a feature in inclusion-body myositis (520). Inclusion-body myositis is differentiated from limb-girdle muscular dystrophy by pattern of weakness and characteristic histopathological changes including rimmed vacuoles with beta-amyloid inclusions (501; 520).

Various endocrine and toxic myopathies are other treatable disorders that must be differentiated from limb-girdle muscular dystrophy (54; 461). Myopathies due to treatment with chloroquine (545) and cholesterol-lowering agents (533) are examples of toxic myopathies, but these compounds are not usually taken at the most common ages of onset for limb-girdle muscular dystrophy.

Diagnostic workup

Blood chemistry. Routine blood counts, serum chemistry, and urinalysis are unhelpful in diagnosing limb-girdle muscular dystrophy. Sedimentation rate may be elevated in inflammatory muscle diseases.

The principal biochemical abnormality in limb-girdle muscular dystrophy, as also in other muscular dystrophies and myopathies, is elevation in the serum concentration of various muscle enzymes (355; 395). Serum creatine kinase is the most widely used enzymatic test for the detection of muscle disease. Creatine kinase is typically elevated in all cases of limb-girdle muscular dystrophy but especially in autosomal recessive severe forms; the creatine kinase levels in severe childhood autosomal recessive muscular dystrophy can be as high as in Duchenne dystrophy (ie, up to over 50 times normal). In other forms of limb-girdle muscular dystrophy, creatine kinase is usually mildly to moderately elevated. In general, creatine kinase levels are less elevated in autosomal dominant forms of slowly progressive limb-girdle muscular dystrophy, and they tend to decrease with the age of the patient and with the duration of the disease. Overall creatine kinase is raised roughly 2x to 350x in recessive cases and is normal to increased 6x in dominant patients (64).

Creatinuria and hypercreatinemia suggest decrease in muscle mass in limb-girdle muscular dystrophy and other dystrophies. Although their value as indicators of muscle disease is limited, they can still be useful in assessing those patients with long-standing limb-girdle muscular dystrophy in whom the serum enzymes are normal.

Electromyography. Electromyographic features in limb-girdle muscular dystrophy are typical of primary muscle disease and are similar to those in other myopathies. The principal findings on needle EMG are short duration, low-amplitude motor unit potentials, an increased proportion of polyphasic potentials, and early recruitment with a full interference pattern in clinically weak muscles. Abnormal spontaneous activity in the form of fibrillation potentials and positive sharp waves can occur in more rapidly progressive limb-girdle muscular dystrophy with prominent muscle fiber necrosis and regeneration, such as severe childhood autosomal recessive muscular dystrophy (214). In the latter setting, fibrillation and positive sharp waves do not result from primary denervation but from segmental necrosis of muscle fibers leading to isolation of a portion of muscle from its myoneural junction. Progressive muscle fibrosis may also result in increased mechanical resistance to insertion of the needle electrode and decreased insertional activity.

Nerve conduction studies, including amplitude of sensory and motor-evoked potentials and conduction velocities of both sensory and motor fibers, are typically normal in limb-girdle muscular dystrophy. However, evoked motor amplitudes can be reduced in muscles with severe atrophy.

Muscle ultrasound. Muscle ultrasound is a safe, noninvasive, and cheap diagnostic tool that can assess muscle thickness and echogenicity to identify atrophic changes and fatty degeneration. It has been helpful in targeted muscle biopsy, but, based on some studies, it can be used as a screening tool for muscular dystrophies. Its main limitation is that it is operator dependent with restricted visualization of deeper structures. In a study by Ibrahim and colleagues, ultrasound images of upper and lower extremities were obtained and visually graded semi-quantitatively using the 4-point Heckmatt scale (232). In muscular dystrophies, there was an increased and diffuse echo from the muscle substance and reduced echo from the bone. The increase in the ultrasound echo correlated with alteration in gross muscle architecture. A total Heckmatt score at a cutoff point greater than two predicted patients with dystrophy, with good (89%) accuracy and with sensitivity and specificity of 100% and 75%, respectively.

Muscle MRI. Muscle MRI can be used to identify patterns of muscle involvement, sparing, and peculiar characteristics of different muscular dystrophies and serves as an easy and fast technique to support a diagnosis. This can aid in the differential and can support pathogenicity of variants of unknown significance. MRI of the thigh and lower leg was found to have high sensitivity for assessing disease progression in LGMD R9 as well as other limb-girdle muscular dystrophies, in particular by detecting global muscle segment fat percentage (419). Table 3 shows MRI findings in different limb-girdle muscular dystrophies.

Table 3. MRI Findings in Different Limb-Girdle Muscular Dystrophies

New nomenclature	Old nomenclature	Gene	Muscles affected	Muscles spared	Characteristic findings
LGMD D1 DNAJB6-related	LGMD 1D	7q36 DNA JB6	Early stages: soleus, adductor magnus, semimembranosus, biceps femoris Later involvement: medial gastrocnemius, adductor longus, vastii	Rectus femoris, lateral gastrocnemius, sartorius, gracilis, anterolateral lower leg muscles (very late involvement)
LGMD D2 TNP03-related	LGMD 1F	7q32.1-q32.2 TNPO3--	Vastus lateralis, sartorius, posterior leg compartment	Gracilis, rectus femoris
LGMD D3 HNRNPDL-related	LGMD 1G	4q21 HNRPDL--	Vastus muscles, posterior thigh compartment	Rectus femoris, adductor longus
LGMD D4 calpain3-related	LGMD 1I	15q CAPN3	Paraspinals, glutei, hamstring, medial gastrocnemius
LGMD D5 collagen 6-related	Bethlem myopathy dominant	21q, 2q COL6A1, COL6A2, COL6A3		Sartorius, gracilis, adductor longus	Vastus lateralis: rim of hyperintensoty at the periphery Rectus femoris: hyperintensity at the center of the muscles (central shadow) Rim of hyperintensity between the soleus and gastrocnemius
LGMD R1 calpain3-related	LGMD 2A	15q CAPN3	Gluteus maximus, semimembranosus, biceps femoris, adductors, soleus, medial gastrocnemius, serratus anterior, lumbar erector spinae	Vastus lateralis, sartorius, gracilis, lateral gastrocnemius
LGMD R2 dysferlin-related	LGMD 2B	2q DYSF	Subscapularis, lumbaris erector spinae, gluteus minimus, tensor fasciae latae, obturator externus, quadriceps, semimembranosus, semitendinosus, biceps femori, hip adductors, all leg muscles (except popliteus)	Head and cervical muscules, levator scapula, trapezius, pectoralis minor, popliteus, piriformis, posterior forearm compartment, transversus abdominis, gracilis, sartorius
LGMD R3 α-sarcoglycan-related	LGMD 2D	17q SGCA	Proximal lower extremity muscles, anterior thigh compartment Quadriceps gradient (more affected proximally than distally), adductor longus medial sparing, hypertrophy of either sartorius or gracilis Paraspinal, pelvic and gluteu muscles can be affected in mild cases	Relative sparing of leg muscles until late stages Tensor fasciae latae, tibialis posterior, flexor digitorum longus
LGMD R4 β-sarcoglycan-related	LGMD 2E	4q SGCB	Proximal lower extremity muscles, anterior thigh compartment Quadriceps gradient (more affected proximally than distally), adductor longus medial sparing, hypertrophy of either sartorius or gracilis	Relative sparing of leg muscles until late stages Tensor fasciae latae, tibialis posterior, flexor digitorum longus
LGMD R5 γ -sarcoglycan-related	LGMD 2C	13q SGCG	Proximal lower extremity muscles, anterior thigh compartment Quadriceps gradient (more affected proximally than distally), adductor longus medial sparing, hypertrophy of either sartorius or gracilis	Relative sparing of leg muscles until late stages Tensor fasciae latae, tibialis posterior, flexor digitorum longus
LGMD R6 δ-sarcoglycan-related	LGMD 2F	5q SGCD	Proximal lower extremity muscles, anterior thigh compartment Quadriceps gradient (more affected proximally than distally), adductor longus medial sparing, hypertrophy of either sartorius or gracilis	Relative sparing of leg muscles until late stages Tensor fasciae latae, tibialis posterior, flexor digitorum longus
LGMD R7 telethonin-related	LGMD 2G	17q TCAP	Glutei, hip, thigh and tibialis anterior	Sartorius
LGMD R8 TRIM 32-related	LGMD 2H	9q TRIM32	Posterior thigh and posterior leg compartments	Flexor digitorum longus, flexor hallucis longus, tibialis posterior
LGMD R9 FKRP-related	LGMD 2I	19q FKRP	Glutei (maximus> medius), adductors, biceps femoris, vastus intermedius, vastus lateralis, medial gastrocnemius, soleus Mild cases with only adductor magnus involvement	Rectus femoris (late), sartorius, gracilis, tibialis anterior
LGMD R10 titin-related	LGMD 2J	2q TTN	Anterior leg and thigh compartment
LGMD R11 POMT1-related	LGMD 2K	9q34.1 POMT1
LGMD R12 anoctamin5-related	LGMD 2L	11p ANO5	Gluteus minimus, posterior thigh and posterior leg compartments Isolated semitendinosus involvement	Gracilis
LGMD R13 Fukutin-related	LGMD 2M	9q FKTN
LGMD R14 POMT2-related	LGMD 2N	1p POMT2	Hamstring, paraspinals, glutei, posterior leg compartment
LGMD R15 POMGnT1-related	LGMD 2O	14q POMGnT1
LGMD R16 α-dystroglycan-related	LGMD 2P	3p DAG1
LGMD R17 plectin-related	LGMD 2Q	8q PLEC	Posterior leg compartment
LGMD R18 TRAPPC11-related	LGMD 2S	4q TRAPPC11
LGMD R19 GMPPB-related	LGMD 2T	3p GMPPB	Paraspinal and hamstring
LGMD R20 ISPD-related	LGMD 2U	7p ISPD
LGMD R21 POGLUT1-related	LGMD 2Z	3q POGLUT1			Early fatty replacement of internal regions of thigh muscles that spared the external area
LGMD R22 collagen 6-related	Bethlem myopathy recessive	21q, 2q COL6A1, COL6A2, COL6A3
LGMD R23 laminin α2-related	Laminin α2-related muscular dystrophy	LAMA2	Adductor magnus, biceps femoris	Sartorius, gracilis	Hyperintensity in periphery of thigh muscles
LGMD R24 POMGNT2-related	POMGNT2-related muscular dystrophy	POMGnT2
LGMD R26 Popeye domain-containing protein 3 related	Popeye domain-containing protein 3 related	POPDC3	Vastus lateralis, gastrocnemius, medial head		Fatty replacement in affected muscles and paraspinals
Reclassified conditions (no longer included in the new LGMD classification)
Myofibrillar myopathy	LGMD 1A	5q MYOT	Soleus, medial gastrocnemius, sartorius
Emery–Dreifuss muscular dystrophy (EDMD)	LGMD 1B	1q LMNA	Adductor magnus, semimembranosus, short and long head of the biceps femoris, quadriceps (except rectus femoris)	Gracilis, sartorius, rectus femoris
Rippling muscle disease	LGMD 1C	3p CAV3	Rectus femoris, semitendinosus, gastrocnemius		Peripheral hyperintensity in rectus femoris
Myofibrillar myopathy	LGMD 1E	6q DES	Semitendinosus, sartorius, gracilis, peroneal muscles, tibialis anterior
?	LGMD 1H	Not confirmed
Myofibrillar myopathy	LGMD 2R	6q DES
Pompe disease	LGMD 2V	17q GAA
PINCH-2 related myopathy	LGMD 2W	PINCH2
BVES related myopathy	LGMD 2X	BVES
TOR1AIP1 related myopathy	LGMD 2Y	1q TOR1AIP1

Muscle biopsy. Muscle biopsy of a clinically involved but not yet end-stage muscle in limb-girdle muscular dystrophy cases shows typical findings of primary muscle disease with additional signs pointing to a dystrophy. In most patients, the EMG and muscle biopsy findings are concordant (62). Muscle biopsy samples in general are characterized by a number of nonspecific pathological changes similar to those observed in other dystrophies (391). The severity of pathological changes and the tempo of progression are highly variable. Although the changes in the severe childhood form of autosomal recessive limb-girdle muscular dystrophy are similar to those observed in Duchenne patients, the findings in other forms are not as severe. No morphological features with routine histological stains differentiate one limb-girdle dystrophy from another or from other muscular dystrophies (391; 12).

In cross-section, there is a wide spectrum of muscle fiber sizes, ranging from large, hypertrophied fibers to small atrophic fibers in the same fascicle. Individual muscle fibers typically lose their polygonal shape and become rounded, and the nuclei often occupy central areas. The endomysial connective tissue increases with progression of the disease process. A nonspecific but wide range of degenerative changes include fiber splitting, ring-fibers, and lobulated fibers (41), and individual muscle fibers showing hyalinization, vacuolation, necrosis, and phagocytosis may be observed. Regenerating fibers with prominent nucleoli and basophilic sarcoplasm are often seen. There are occasional mononuclear cellular infiltrates near necrotic muscle fibers notably in the dysferlinopathies, but these are neither prominent nor widespread. Degenerating muscle fibers are eventually replaced by adipose or connective tissue. Some of these pathologic features have been seen in one syndrome more than another, but as with many of the clinical features there is too much overlap to rely on any one as a differentiating feature. Although next-generation sequencing is typically the diagnostic method of choice, muscle biopsy still has a role when the genetic diagnosis is complicated as it can help to assess changes to dystrophin complex properties (109).

Histochemical studies disclose nonspecific changes typical of primary muscle disease (96). Whorled fibers, moth-eaten fibers, and lobulated fibers, if present, are better appreciated when oxidative enzymes are used.

There have been few ultrastructural studies of muscle in limb-girdle muscular dystrophy. Most authors have described findings in "muscular dystrophy" as a group without specifying the type of dystrophy (391; 436). Most reported ultrastructural reactions involve the myofibrils and mitochondria and are nonspecific. Focal myofibrillar degeneration and Z-disk streaming are common in limb-girdle muscular dystrophies, as in other muscular dystrophies. Sarcoplasmic masses, autophagic vacuoles, small focal collections of glycogen, and scattered lipid droplets are also occasionally encountered.

Immunostaining for specific proteins, however, is highly useful, especially with the sarcoglycanopathies, and has improved the ability to diagnose some of the specific disorders short of molecular genetics. Staining for dystrophin in both males and females is essential to exclude affected individuals and manifesting carriers. Although adhalin (alpha-sarcoglycan) is a marker of the dystrophin-associated glycoprotein complex, specific antibodies for each subunit are available. Staining for merosin is useful in congenital cases. Antibodies for other proteins are increasingly available but vary between neuropathological centers.

Specific genetic tests. All known forms, including those only linked to chromosomal regions, can be theoretically screened by molecular genetics; however, some genes are more complex than others. For example, in excess of 500 mutations of the calpain-3 gene are known even though most cases are caused by a single mutation (188; 141). The most current information can be found on this frequently updated website: Genetic Testing Registry. However, many cases and families diagnosed as limb-girdle muscular dystrophy on clinical grounds remain unconfirmed and unclassified.

Currently, the best approach to obtain a genetic diagnosis in cases of limb-girdle muscular dystrophy is the use of next-generation sequencing so that patients can be screened using custom DNA capture via neuromuscular disease-related gene panels or by whole exome sequencing (180). This approach increases diagnostic yield and leads to discovery of new genes and phenotypes (266; 345; 444; 209; 417; 559). It is worth mentioning that repeat disorders such as fascioscapulohumeral muscular dystrophy or myotonic dystrophy should be excluded first before doing next-generation sequencing. Although this approach increases diagnostic yield, there will still be a group of patients who remain undiagnosed (from 25% to 70%). In a study, a genetic diagnosis was obtained in 53% of families using a multigene next generation sequencing panel as a first-tier approach in the investigation of muscular dystrophy (547). If next-generation sequencing is negative or unclear, further work up with neurophysiological and imaging studies, respiratory and cardiac investigations, and biopsy and protein immunoanalysis come into play. It is expected that with improvement of genetic testing and patient registries, more mutations and new phenotypes of known pathogenic genes will be described.

Management

No specific therapy to halt or delay progression of the primary muscle disease is presently known for any of the limb-girdle muscular dystrophies. Glucocorticoids have shown to improve muscle strength in Duchenne and Becker muscular dystrophy; however, no improvement has been observed in most patients with limb-girdle muscular dystrophy when treated with a 6-month course of prednisone (231). However, there have been small reports of improvement in LGMD R6 and LGMD R9. In LGMD R2, dysferlin-related, a case report of a patient using 15 mg prednisone orally in alternate days showed an increment of elbow and knee flexion strength over the follow-up time of 17 years (487). In mouse models with mutations for LGMD R2 and LGMD R5, they found that use of steroids was associated with better sarcolemmal repair. This effect was most prominent for vamorolone, however, the relationship between anti-inflammatory and functional improvement is still not completely clear as it was not examined in this study. Two siblings with LGMD R4 were treated with 0.9 mg/kg/day of deflazacort and demonstrated improvement in muscle testing at 22 months. Prednisone has also been used with some clinical benefit in LGMD R6. However, for LGMD R2, deflazacort was used for daily therapy and was associated with decreased muscle strength. There are some ongoing trials utilizing daily deflazacort in LGMD R9 with 4-stair climb times (4SCT) as a primary outcome and another study is using weekly prednisone in patients with all LGMD forms, measuring safety and muscle function improvement as the outcomes (413).

In LGMD R9 and LGMD R1, low-intensity training resulted in an increase in endurance and strength in a small study (483). Antigravity training improved walking capacity and postural balance in LGMD R9 (39). A mouse model for LGMD R2 compared the effects of concentric muscle contractions to those of eccentric contractions and found that concentric training conferred exercise benefits without inducing the damage that eccentric training did (33).

Myostatin inhibition therapy was attempted for patients with LGMD R1 that resulted in muscle hypertrophy, actually creating a loss of oxidative capacity and unfortunately no increase in muscle function or exercise tolerance (274). In dysferlin deficient mice, N-acetylcysteine supplementation reduced oxidative damage to muscles and also improved the animals’ grip strength, indicating that this antioxidant could be of benefit to patients with LGMD R2 (172).

In one patient with dominantly inherited calpainopathy (c.643_663del21 mutation), an open label treatment with low-dose recombinant human growth hormone (somatropin) over a period of 4.5 years improved muscle strength measure with dynamometer and stabilized walking ability (measured by 6-minute walk test). This suggests a possible role of human growth hormone in limb-girdle muscular dystrophy treatment (409).

Cell based therapeutic approaches could be another option that may prove suitable in the near future. Stem cell transplantation promoted muscle regeneration in a mouse model by returning alpha DG glycosylation to mice who were FKRP deficient, mimicking the LGMD R9 state (15). Another study used human induced pluripotent stem cells to generate healthy muscle cells and neurons in vitro in the presence of the LGMD R1 mutation (325).

Additionally, small molecule inhibition has been investigated for mice with LGMD D1 states. Treatment of these mice with an inhibitor for the DNAJ-HSP70 complex remobilized HSP70 away from the Z disc and corrected myopathology (37). A phase 1 clinical trial with safety measurement for the antiapoptotic compound omigapil was conducted for LGMD R23, characterized by LAMA2 gene mutations (439). For the sarcoglycanopathies (LGMD R3, 4, 5, 6), CFTR correctors successfully removed defective sarcoglycan-complexes in vitro when the mutations were missense (74).

The rapidly advancing insights into the mechanisms of muscle cell death have opened the way for genetic interventions. Strategies include the use of viral vectors (such as adeno-associated virus vector) to deliver the mutated genes.

For LGMD R5, an experiment using vivo-PMOs (anti-sense oligonucleotide) mediated-exon skipping demonstrated successfully expressed sarcoglycan proteins; however, these studies have not been performed yet in therapeutic trials. A clinical trial has been done for AAV (adeno-associated viral vectors) gene transfer therapy showing no adverse effects for these patients and also that there was a dose-dependent response for how much gamma sarcoglycan was expressed (90).

In LGMD R4 (LGMD2E), three children treated with systemic administration of the investigational gene therapy MYO-101 (scAAVrh74.MHCK7.hSGCB) showed increment of beta-sarcoglycan expression in muscle (mean of 51% of positive fibers exceeding the predefined measure for success of 20%) at 2 months with 90% reduction of creatine kinase. The vector promoter (MHCK7) was chosen for its ability to generate significant protein levels in the heart, which can be affected in LGMD2E patients. A phase I/IIa study cohort of six patients is ongoing with an estimated completion date at the end of 2020.

In LGMD R3 (LGMD2D), a phase I/IIa study of alpha-sarcoglycan gene delivery using a self-complementary adeno-associated virus vector, scAAVrh74.tMCK.hSGCA (MYO-102), was done using isolated limb infusion. One nonambulant adult received the treatment in one leg. Once confirmed to be safe, five ambulatory children received the drug in both legs. They were followed up for a total of 2 years. Following treatment, four of the children had increased levels of alpha-sarcoglycan in muscles. Two of the children had changes in thigh muscle strength. However, the 6-minute walk test worsened or remained the same in all subjects (327). The authors explained that lack of delivery to more proximal hip muscles affected the ability to improve ambulation, so a trial of systemic administration was conducted on a mouse model in 2020, which led to expression of the deficient protein (alpha sargoclycan) without findings of vector toxicity and conferred some benefit to the mice (191).

In LGMD R2 (LGMD2B) patients, a phase I study of intramuscular administration of dysferlin gene using AAVrh.74.Dysferlin.DV (dual vectors) (MYO-201) was completed. In this approach, two viral vectors will be administered to reconstitute the full-length dysferlin gene. This approach was previously successful in dysferlin-deficient mice (467; 406).

For calpainopathy LGMD (types R1 and D4), recombinant AAV-mediated calpain 3 transfer has demonstrated improvement in animal studies, but gene therapy clinical trials are yet to occur (315). An animal study also demonstrated AAV CAPN3 gene therapy in a mouse that resulted in significant, robust improvement in functional outcomes and muscle physiology independent of age (432).

Other gene replacement therapies in the horizon, all using AAV vectors, include MYO-103 (LGMD2C or gamma-sarcoglycan), MYO-301 (LGM2L or ANO-5), and LGMD2A (CAPN3).

In a mouse model of LGMD R9, long-term ribitol treatment has shown improved muscle pathology and function and increased lifespan without serious side effects (551). In addition to the 2017 reclassification, Barton and colleagues suggested the consideration of functional clusters for limb-girdle muscle dystrophies: dystroglycanopathies, mechanical signaling defects, and mitochondrial dysfunction, a grouping that could eventually inform potential therapies that are not just gene therapies (23). The glycosylation cluster includes LGMD R9, R11, R13, R14, R19, R20, R24, for which experimental treatments are being investigated on the effects of augmenting glycosylation through overexpression of LARGE, or ISPD, which are dystroglycanopathy genes. The mechanical signaling defects cluster includes LGMD R1, R2, R5, R6, for which treatments with proteins modulating the phosphorylation state of sarcoglycans could be of benefit, as could suppression of P-p38 vs disinhibition of p38 activity to allow for proper tuning of this signaling pathway when an aberrancy exists. The mitochondrial dysfunction group includes LGMD R1, R2, R3, R5, R6, for which treatments could involve mitochondrial-targeting strategies (23).

In particular, treatment with the AMPMP compound for an in vivo model of LGMD R1 activates signaling of defective CaMKIIbeta, which lends support for this as a potential future therapeutic option (298). Similarly, recombinant human galectin-1, a soluble carbohydrate binding protein was found to increase myogenic potential in dysferlin deficient cells, suggesting a potential therapeutic target for LGMD R2 (511). Another study used GW501516 (a PPARδ agonist) to improve mitochondrial biogenesis in vivo in 7-month-old calpain-3-deficient mice (241). This treatment improved satellite cell activity and sarcolemmal repair, reduced serum CK levels, and reduced muscle fatiguability, lending this is a potential therapy for LGMD R1.

Nonspecific therapeutic measures to improve residual muscle strength and to help preserve optimum function are advocated. Primary goals are to prevent contractures and deformities and to maintain upright posture and ambulation as long as possible. Passive stretching exercises, judicious use of orthopedic procedures and bracing, and active exercises against graded resistance are all important measures in the care of these disorders.

Genetic counseling is always a sensitive issue, particularly in families with limb-girdle muscular dystrophy. Counseling should take into account the possibility of considerable interfamilial and intrafamilial variation in the severity of the disease and possible founder effects. It can be difficult to convince family members that the risk of having a relatively severely affected child may be as high for mildly as for severely affected parents.

The cardiopulmonary manifestations are managed by conventional means. Early and severe diaphragmatic involvement may lead to chronic alveolar hypoventilation characterized by early-morning headache, excessive daytime sleepiness, fatigue, and altered cognition. Assisted nocturnal ventilation may provide sustained relief from these symptoms. Periodic assessment of respiratory function is important for early recognition and timely management of respiratory insufficiency. Cardiac evaluation should be done for patients with myotylin, lamin, desmin, DNA JB6, sarcoglycans, FKRP, titin, TRIM32, telethonin, POMT1, POMT2, fukutin, PMGnT1, and dystroglycan mutations or if patient has abnormal EKG, syncope, near-syncope, or palpitations (361). In particular, for the c.826C>A variant for those with FKRP mutations (LGMD R9), Libell and colleagues have suggested that homozygotes have echocardiogram monitoring every 2 to 3 years in childhood/adulthood whereas other FKRP genotypes should undergo this annually as cardiomyopathy tends to develop earlier in these groups (290). A study suggested surveillance with cardiac MR for limb-girdle muscular dystrophy patients, as MR can detect cardiac involvement earlier than echo or EKG, especially because patients with limb-girdle muscular dystrophy whose cardiac function is affected may manifest with preserved LV volume and LVEF despite dilated cardiomyopathy (435). Characteristic findings can include subepicardial late gadolinium enhancement in the inferior and inferolateral walls or mid-myocardial late gadolinium enhancement in the basal septum.

Pashun and associates have estimated that LGMD R4 is associated with cardiomyopathy in approximately 50% to 67% of patients, in whom intramyocardial fat infiltration, ischemic events, and early cardiac death can occur (388). Although there are no formally established guidelines yet for the routine surveillance of these patients, early cardiac imaging is important and ECG, Holter monitoring, and echocardiogram can be considered at a frequency of every 2 to 5 years to prevent complications associated with the cardiac damage that commonly occurs in LGMD R4 patients. Cardiac MR is the gold standard for myocardial tissue characterization to demonstrate scarring; however, cardiac CT can be used with more ease and can also detect intramyocardial fat, so it could, therefore, serve as an important means of surveillance as well. LGMD R4 is associated with abnormalities of the sarcoglycan complex in vascular smooth muscle and mouse models have revealed a role for verapamil initiation to alleviate vascular constriction and potentially prevent cardiomyopathy before it occurs in these patients (388).

Regarding swallowing function, patients with dysphagia, frequent aspiration, or weight loss should be referred for swallowing evaluation or gastroenterology evaluation to assess and manage swallowing function and aspiration risk and to teach patients techniques for safe and effective swallowing (eg, chin tuck maneuver or altered food consistencies) as well as to consider placement of a gastrostomy or jejunostomy tube for nutritional support.

Muscular dystrophy patients with musculoskeletal spine deformities should be evaluated by an orthopedic spine surgeon for monitoring and surgical intervention if it is deemed necessary in order to maintain normal posture, assist mobility, maintain cardiopulmonary function, and optimize quality of life.

As a whole, patients with muscular dystrophy should have access to a multidisciplinary clinic where they can be evaluated by multiple specialties (physical therapy, occupational therapy, respiratory therapy, speech and swallowing therapy, cardiology, pulmonology, orthopedics, and genetics) in order to receive efficient and effective long-term care.

Attending to the emotional needs of patients with disabling muscular dystrophy is an exceedingly important aspect of the overall management.

Special considerations

Pregnancy

Some forms of disease have been successfully diagnosed prenatally through chorionic villus sampling.

A study examined pregnancy outcomes for women with LGMD R9 (289). They had higher rates of labor induction and were found to be significantly more likely to require assisted vaginal delivery and some also experienced a progression of weakness during their pregnancy, at times without a return to their prior baseline.

Anesthesia

Patients with advanced limb-girdle muscular dystrophy and a compromised cardiopulmonary status may be at increased risk for perioperative morbidity and mortality.