The muscle pathology in our study of DNAJB6 mutated LGMD1D shows distinct findings that should provide clues for a correct diagnostic approach even in a sporadic patient without known family history. On light microscopy the most common finding is the presence of early stage myofibrillar disintegration with small myofibrillar aggregates and rimmed vacuolated fibers. On immunohistochemistry many proteins are found in the aggregates, and the rimmed vacuoles are reactive for several proteins associated with autophagy. The light microscopy findings are in general similar to MFMs. However, the myofibrillar aggregates are commonly distributed in scattered fibers and are smaller than in a typical MFM. Only in one biopsy large and pleomorphic aggregates in groups of fibers unevenly distributed across the fascicles more characteristic of a typical MFM were seen. In addition, rubbed-out fibers commonly found in MFMs such as desminopathies and alphaB-crystallinopathies are not a common feature in LGMD1D.
Secondary inflammatory changes that often may occur in genetic myopathies were observed in approximately half of the DNAJB6 mutant biopsies. However, the endomysial inflammatory cells were few in number without larger infiltrates and no invasion in non-necrotic fibers was observed. In addition, there was no significant expression of MHC HLA Class I, thus clearly preventing any confusion with sporadic inclusion body myositis (s-IBM) despite the rimmed vacuolation. In addition, no significant numbers of cytochrome oxidase-negative muscle fibers or ragged red fibers were present. Clinically, the involvement of hamstrings was always more severe than the quadriceps and without the finger flexor weakness as with s-IBM.
The ultrastructural findings in myofibrillar myopathies may show some differences in the genetically different myopathies . Compared to myotilinopathy the changes in LGMD1D show less frequently tubulofilamentous bundles and no basement membrane thickening, described in myotilinopathy . In zaspopathy filamentous accumulations in bundles are frequent and intrasarcoplasmic rods common, but were not observed in LGMD1D. Desminopathies characteristically show cytoplasmic electrondense reticular granulofilamentous accumulations, in addition to areas with sarcomere disorganization and Z-disk alterations. Areas of amyloid-like material often adjacent to granulofilamentous inclusions or in vacuolated fibers can also be found in desminopathy, as well as other myofibrillar myopathies [8, 16]. Reticular desmin aggregates or amyloid deposits were not found ultrastructurally in the studied LGMD1D biopsies. The large complexes of electrodense granulofilamentous accumulations and sandwich formations in desminopathy and αB-crystallinopathy and filamentous bundles and extensive Z-disk alterations in myotilinopathy and zaspopathy were not observed ultrastucturally in LGMD1D.
The autophagic degenerative component of the pathology in LGMD1D with the basic features of rimmed vacuoles on light microscopy and disorganized myofibrillar structures, Z-line dispersion, and dilatation of sarcoplasmic reticulum into vacuolar formations on ultrastructure are not particularly different compared to the similar pathology of the other myofibrillar myopathies or s-IBM. However, eosinophilic inclusions in the rimmed vacuoles that occasionally may be observed in s-IBM were rare in the LGMD1D samples.
LGMD1D is caused by mutations in DNAJB6 gene . DNAJB6 belongs to the evolutionarily conserved DNAJ / HSP40 family of proteins, which regulate molecular chaperone activity by stimulating ATPase activity . DNAJB6 is expressed in many tissues  with its highest expression in brain. Its association to skeletal muscle disease was shown in 2012 . Our previous studies suggested DNAJB6 involvement in the CASA pathway, a major mechanism in protein re-cycling and turnover for the maintenance of Z-disc sarcomeric integrity. We have in our previous work found that DNAJB6 interacts with several members of CASA complex: BAG3, HSPA8, STUB1 and HSPB8 . In this study we also demonstrate in patient muscle biopsies the involvement of these proteins, thus confirming the results shown with the F93I mutation . The dominant toxic effect of the mutant F93L decreases the anti-aggregational effect of DNAJB6 and makes the whole chaperonal complex in which DNAJB6 is involved less effective, leading to protein aggregations and secondary autophagic abnormalities with rimmed vacuolar pathology. Because of the defective DNAJB6 function Z-disc components are disturbed and accumulate first (myotilin, desmin, αB-crystallin, BAG3) followed by defective autophagy which can be seen as staining of rimmed vacuolar markers LC3, VCP, TDP-43, SMI-31 and p62 increasing with disease duration. These findings, when observed in the clinical diagnostic praxis, in particular the early changes of smaller myofibrillar aggregates in normal sized fibers should direct the further diagnostic efforts towards DNAJB6 and LGMD1D disease.
Exceptionally, LGMD1B has been reported to cause myofibrillar changes similar to LGMD1D, however without rimmed vacuolar pathology .
Histopathology of LGMD1D is different from the other more prevalent autosomal dominant limb-girdle muscular dystrophies; markedly different from the usually nonspecific pathology in LGMD1B and LGMD1C and also distinct from the more closely related LGMD1A myotilinopathy.