gene consists of 32 exons and is ubiquitously expressed, including in skeletal muscle, skin and heart [1
]. Plectin is a large protein ranging from 4684 amino acids in the canonical form to 3447 amino acids in the smallest stable isoform. It has a unique molecular structure: the N-terminal domain is encoded by multiple short exons, whereas the central-rod domain and the C-terminal domain are encoded by single large exons. Exons 2-32 are constant, but eight different-sized forms of exon 1 exist, which undergo alternative splicing into exon 2. At least eight different plectin isoforms (1, 1a, 1b, 1c, 1d, 1e, 1f, and 1g) resulting from the different splicing of exon 1 have been identified [2
]. These isoforms have various lengths, localizations, interacting partners, and specific functions. For instance, plectin 1a is localized to the outer nuclear/endoplasmic reticulum membrane system, plectin 1b to the mitochondria and plectin 1d at the Z-disks [3
]. Plectin 1f, in particular in striated muscle, has been shown to interact with components of the dystrophin-glycoprotein complex [4
] but also to play an important role in the neuromuscular junction (NMJ) where it interacts directly with rapsyn [5
]. Plectin stabilizes the desmin cytoskeleton and interacts with its three main components: actin microfilaments, microtubules, and intermediate filaments. Other tissue specific roles depend on the specific splicing isoform and the numerous binding partners, such as α-actinin, vimentin, keratin, and desmin, which interact with different regions of the plectin protein [3
Mutations in PLEC
are associated with multiple phenotypes depending on the location of the mutation and isoform affected. Epidermolysis bullosa simplex with muscular dystrophy [EBS-MD (MIM #226670)] is caused by recessive mutations [7
], mostly nonsense, out-of-frame insertions or deletions within exon 31 and 32, leading to premature protein termination. Individuals with EBS-MD show severe skin blistering, sometimes accompanied by skin atrophy, alopecia, and nail dystrophy, with first symptoms usually starting in infancy and even leading to premature death in childhood. The EBS-MD phenotype shows high clinical variability with a few individuals also presenting with myasthenic symptoms and additional features, such as anemia and cardiomyopathy [9
]. Additionally, a phenotype of recessive limb-girdle muscular dystrophy, LGMD R17 plectin-related (MIM #613723, previously known as LGMD 2Q), was reported [10
]. LGMD R17 is associated so far with only recessive truncating mutations located in exon 1f, and manifests with muscle weakness without any skin involvement. This phenotype was first described in three consanguineous Turkish families carrying a c.1_9del mutation and presenting with young adult/childhood onset muscle weakness and dystrophic changes in the muscle biopsy, without skin involvement or myasthenic features [10
The association of plectin deficiency with muscle weakness is already well characterized. However, the role of plectin deficiency in patients with features of a congenital myasthenic syndrome is still not well understood and is only based on a few case reports. Herein, we describe four individuals with LGMD, easy fatigability, ptosis, and clinical responsiveness to acetylcholinesterase inhibitors and salbutamol, features commonly associated with myasthenic syndromes. They all carry the known c.1_9del PLEC mutation in homozygosity. All four patients included in this study originate from the same region near the Black Sea, in Turkey. We also identified a shared haplotype in three individuals suggesting a common origin of the mutation. Patients with myasthenic symptoms of unknown origin should be tested for mutations in the PLEC gene.
We present four patients from unrelated families carrying the same PLEC mutation and presenting with limb-girdle weakness and myasthenic symptoms. All four patients had shoulder and pelvic girdle weakness with variable degree of fatigable ptosis, nasal speech, and swallowing difficulties. Paraspinal muscle weakness was also very prominent in our patients. All complained of easy fatigability and muscle cramps. The age of onset of symptoms varied from the infantile period with delayed motor milestones to early adulthood. They all had elevated serum CK levels and a high jitter on RNS. All patients benefited from pyridostigmine and salbutamol treatment.
The most common neuromuscular phenotype associated with mutations in PLEC
is epidermolysis bullosa simplex with muscular dystrophy (EBS-MD; MIM #226670). Symptoms include skin blistering and ocular and neuromuscular involvement later in life [9
]. The disease is inherited in an autosomal recessive manner and is associated with a dissociation of desmin filaments and, consequently, protein aggregates positive for desmin, syncoilin, and synemin accumulate in muscle fibers. Myofibrillar changes can be observed histologically and secondary mitochondrial pathology has been described [12
]. Additionally, some patients present with systemic involvement (e.g., gastroenterological, dental, urogenital and cardiac) [13
]. A few individuals with EBS-MD show myasthenic features, such as fatigability, ptosis, dysphagia or nasal speech [7
]. However, these features were not investigated systematically and additional tests, such as SF-EMG had previously only been performed in a single case. The data from our four patients showed that the myasthenic phenotype may be more common in patients with PLEC
mutations but may be overlooked when the clinical presentation is only mild. All EBS-MD mutations identified so far, apart from one, were in exons common to all plectin isoforms, and in the majority of cases lead to premature termination of protein translation. Other phenotypes associated with PLEC
mutations are autosomal recessively inherited EBS with pyloric atresia (EBS-PA; MIM #612138) [18
] and autosomal dominantly inherited EB simplex Ogna type (EBS-OG; MIM #131950) [19
]. These diseases are associated mainly with C-domain PLEC
In addition, patients with recessive PLEC
mutations and a LGMD phenotype, but without any skin involvement (LGMD R17, previously known as LGMD 2Q), have also been reported. The phenotype was first described in three consanguineous Turkish families (six affected individuals in total) carrying the same homozygous c.1_9del mutation identified in the patients described here. This 9 bp deletion contains the ATG initiation codon located in exon 1f, and results in impaired translation of the plectin 1f isoform exclusively [10
]. More recently, another truncating variant in exon 1f (p.Glu20Ter), also resulting in a LGMD R17 phenotype, has been reported [20
The four families homozygous for the c.1_9del mutation that are characterized in this manuscript show many common features with the previously described families with LGMD R17, and none of the patients showed skin or nail involvement. However, there were some important differences between the patients described in this manuscript and those reported by Gundesli et al. [10
] (Table 1
). All patients displayed clear myasthenic symptoms, such as ptosis, fatigability, nasal speech, and a positive clinical response to the treatment with pyridostigmine and salbutamol. Additionally, one patient’s onset was in adulthood with a completely asymptomatic childhood period. All four patients described in this manuscript had paraspinal muscle weakness and calf hypertrophy. It is possible, however, that the myasthenic features might indeed not have been manifested in patients that were even younger than the ones we report. Alternatively, the more severe muscular dystrophy phenotype seen in the c.1_9del patients reported by Gundesli et al. might have masked the myasthenic symptoms.
Our patients come from unrelated consanguineous families from the Black Sea region in Turkey, as were the individuals described by Gundesli et al. who identified –using polymorphic DNA markers—a shared haplotype of ~2.3 Mb upstream of the PLEC
variant in five of their patients [10
]. Our haplotype analysis using WES data for three of our patients confirms the founder effect for the PLEC
variant in the Turkish population but also delineates the breakpoints and size of the haplotype more clearly at 3.8 Mb.
Up until now, myasthenic phenotypes were frequently associated with mutations in the C-domain of PLEC.
However, the cases described here carry the c.1_9del mutation, located in exon 1 of the plectin 1f isoform, and no other rare damaging variants in the PLEC
actin binding domain have been detected (Figure 5
). Plectin 1f plays an important role in the NMJ and interacts directly with rapsyn [5
]. Our findings support the hypothesis of a crucial role of plectin 1f isoform in the NMJ and in the pathogenesis of myasthenic features. Although we only found a decremental response of 10% or more on RNS in two of our patients, the other two patients had severe weakness due to the muscular dystrophy, which might have masked the dysfunction of the NMJ. The patients who had less severe weakness and more prominent decremental response on RNS, had the most benefit from treatment. Early diagnosis and treatment of these patients may be more effective and lead to an improved quality of life.
To the best of our knowledge, only a few cases of EBS-MD with myasthenia have been described so far (Table 2
). However, myasthenic features in EBS-MD patients may be missed if they are not investigated systematically and no additional tests such as SF-EMG are performed.
Plectin is a structural scaffolding protein concentrated at sites of mechanical stress and in the Z-disc. In PLEC
patients with myasthenic features, neuromuscular junctions showed abnormal morphology with disrupted structures and degeneration [5
]. At the molecular level, it was shown that plectin 1f plays an important role in the stability of the NMJ [4
]. Electron microscopy analysis of muscle biopsies of patients with start codon loss in 1f isoform showed empty spaces between the sarcolemma and the contractile elements [10
]. It was suggested that PLEC 1f interacts with desmin intermediate filaments (IFs) in the postsynaptic domain of the NMJ and plays a central role in their stability by directly interacting with rapsyn. Staining performed specifically for the PLEC 1f isoform in the NMJ showed co-localization with acetylcholine receptors (AChRs) [5
] and in plectin-deficient myoblasts, AChRs are highly mobile and cannot form stable clusters. PLEC 1f also interacts with desmin and studies in PLEC 1f deficient myoblasts showed that without such a linkage the structural network of NMJ collapses [5
]. Conditional plectin knockout mice (Pax7-Cre/cKO) with postsynaptic absence of plectin, resulted in a phenotype similar to EBS-MD-MyS, with severe lack of coordination, reduced motility, and shortened life span. In this model, uncoupling of AChRs from IFs resulted in severe disorganization and fragmentation of the NMJ and depletion of AChRs. Instead, presynaptic lack of plectin did not cause reduced muscle strength in mice lacking P1c plectin isoform [5
Phenotypes associated with PLEC
mutations are highly variable, which may reflect the multiple binding partners of the plectin protein. The cause of this clinical variability is not yet fully understood but could be explained by the location of the mutations in different domains and various isoforms [27
]. As many patients with plectin deficiency have not been thoroughly investigated for NMJ defects, it may be that myasthenic features have been underestimated. Our cases carrying the c.1_9del PLEC
mutation broaden the LGMD R17 phenotype with onset in adulthood along with extraocular, facial, and tongue involvement. Individuals with PLEC
mutations should be carefully examined for myasthenic features and SF-EMG is crucial to confirm the diagnosis.