The Pathomechanism And Genotype-Phenotype Relationship In Hereditary Inclusion Body Myopathy

IntroductionHereditary inclusion body myopathy (HIBM) is an autosomal recessive inherited disorder which was referred to as quadriceps sparing vacuolar myopathy (QSVR) or distal myopathy with rimmed vacuoles (DMRV) in the past time. QSVR was first reported by Agov in 1984. It is clinically characterized by early involvement of distal muscles of low limbs. Proximal muscles are involved in late stage, but quadriceps is constantly spare. Pathological findings include rimmed vacuole formation and intranuclear filamentous inclusions. In 2001, Eisenberge identified UDP-N-acetylglucosamine2- epimerase/acetylmannosamine kinase (GNE) gene, which was localized on 9P12-13. All QSVR patients reported in Middle Easter Jew had homozygous mutation in exon12 which cause a M712T amino acid transition.DMRV was first reported by Nonaka in 1981. This autosomal recessively inherited myopathy was clinically characterized by preferential tibial muscle involvement and, to some extent, sparing quadriceps. Muscle pathology showed rimmed vacuole formation and intranuclear inclusion. Necrotic and regenerative processes, which are common in muscular dystrophy are rare in this disease. In 2002, Kayashima, Tomimitsu and Nishinoconfirmed GNE gene mutation in DMRV. Thus QSVR and DMRV finally proved to be the same allelic diseases and were referred to as HIBM.The pathological characters of HIBM are quite different from muscular dystrophy. Rimmed vacuoles formation and muscle fiber atrophy without marked necrotic and regenerating process were hallmarks of HIBM under light microscope. Rimmed vacuoles are typically detected in small vacuoles lined by many basophilic granules (the rim) on HE staining. Actually these vacuoles are not true empty holes in muscle fibers, but rather artifacts produced during the staining procedure. Ultrastructurally, rimmed vacuoles contain numerous vesicles and myeloid bodies. These components of rimmed vacuoles probably detach easily from glass slid and move to the nearby myofibrils during the staining procedure. Intranuclear filamentous inclusion is the most prominent ultrastructural alteration of HIBM. Taken together with rimmed vacuole formation and nuclear changes, it is suggested that there should be some relationship between these two unique changes. In particular, rimmed vacuoles may be the endproducts of degraded nuclei and their surrounding sacoplasm.Cell deaths were divided into 3 types based on the differences in the ultrastructural morphological features. The type 1 cell death was termed apoptosis, which is characterized by cytoplasmic condensation, nuclear pyknosis, chromatin condensation, DNA fragmentation, membrane blebbing and the formation of membrane bound apoptotic bodies that are rapidly phagocytosed and digested by macrophages or neighboring cells. This process does not induce inflammatory response. Type 2 cell death is characterized by the appearance of numerous cytoplasmic autophagic vacuoles followed by mitochondrial dilation and enlargement of the ER and Golgi apparatus. Other hallmarks of apoptosis such as nuclear pyknosis and membrane blebbing may also occur late in autophagic cell death but are less prevalent. The third type of cell death is necrosis, which is distinguished from the former two types by its lack of autophagosis and lysosomal involvement. Type 3 cell death usually begins with swelling of intracellular organelles followed by the formation of empty space in the cytoplasm, which finally fuse with each other.In HIBM, rimmed vacuole formation was considered to be causative of muscle fiber atrophy. But the pathomechanism of rimmed vacuole is still unknown. Cell death in muscle fibers should appear in focal or segmental pattern since muscle fiber is multinucleated cell formed by fusion of large numbers of myoblasts during myogenesis. It is well known that muscle fiber necrosis is always segmental in most of severe myopathies such as polymyositis and muscular dystrophy. As mentioned above, muscle fiber necrosis and regenerating is very rare in HIBM. Take together with rimmed vacuole contain numerous membranous structures and have a clear demarcating to the surrounding region, we suggested rimmed vacuoles might be endproducts of focal cell death of muscle fibers, including apoptosis and autophagic death.The molecular basis of HIBM has been elucidated. HIBM caused by mutations in the GEN gene which encode a bifunctional enzyme , UDP-N-acetylglucosamine2-epimerase/ acetylmannosamine kinase, catalyzing initial two steps in the biosythesis of sialic acid. A single homozygous missense mutation in exon 12 of the GNE gene is responsible for all HIBM patientsin Middle Eastern Jews, confirming the hypothesis of founder effect in this community. In contrast, about 20 different GNE mutations in either homozygous or compound heterozygous states were identified in Japanese patients. According the literature, Japanese HIBM patients seem to develop severer consequence compared with those in Middle Eastern Jew. Therefore, an analysis of genotype-phenotype correlation and effect of mutations on the kinase and epimerase domain functions is required.ObjectivesTo investigate and confirm apoptotic process in HIBM with different apoptosis-detecting methods.To confirm the possible pathway of apoptotic process in HIBM.To clarify the nature and origin of membranous structures in rimmed vacuoles.To explore the genotype-phenotype correlation of GNE gene mutation in HIBM.MethodsFresh muscle samples from 8 patients with HIBM were prepared for EM observation. We focused on the alterations of nuclei, inclusions and membranous structures, in particular, on the abnormal distribution of nuclear chromatin.In situ TUNEL methods were employed for detecting apoptotic process in frozen muscle samples from HIBM, DMD, PM, ALS and normal control. The percentages of TUNEL positive nuclei were compared in these 5 groups.To test the specificity and sensitivity of TUNEL method employed in this study, we created a classic apoptotic thymus model of Waster rat with prednisolone. Thymus from prednisolone-treated and normal control was prepared for in situ TUNEL method and EM observation. The percentages of TUNEL-positive cells were compared with apoptotic cell detected in semithin sections by light microscope and ultrathin sections under EM.Using immunohistochemical methods we analyzed the expression pattern of apoptotic associated proteins including activated caspase-3, caspase-12, Fas and GRP78.We also investigated the expression patterns of a series of proteins related to membranous organelles including lysosome, autophagosome, endosome, mitochondria, ER, Golgi apparatus and nuclear lamina in HIBM, DMD and normal control.Total DNA was extracted from 22 frozen muscle samples of HIBM. 12 exons of GEN gene were amplified by PCR. We sequenced all the exons and their flanks for detecting gene mutation. Genotype-phenotype correlations were analyzed with summarized clinical and pathological features of all 22 HIBM patients.ResultsBy electron microscope, we found apoptotic nuclei at different stage in HIBM. The apoptotic nuclei of early stage showed dense chromatin filled with supernucleasomes. In contrast, the surrounding contracted components were well preserved. Late-stage apoptotic nuclei showed condensed chromatin, loss of nuclear envelope and finally degraded nuclei. In addition, a huge accumulation of membranous or myeloid structures was noted in rimmed vacuoles.In situ TUNEL stains displayed 0.8 + 0.4% of TUNEL-positive nuclei on average in HIBM muscle samples. But neither early nor late-stage apoptotic nucleus was present in other myopathy or normal muscle control. The number of apoptotic nuclei of prednisolone-treated thymus in semithin sections stained by toludine blue was parellel to positive nuclei detected by TUNEL method. This result reflected that TUNEL method employed in this study was reliable.Activated caspase-3 was expressed in 6.2 + 3.1% of muscle fibers in HIBM, but not in other myopathy or normal control. The percentage of caspase-12-positive fibers was 3.1 + 1.0% in HIBM, which significantly higher than that in other three types of myopathy (PO.001). In normal control, there is no caspase-12-positive fiber. 5.2 + 2.4% muscle fibers showed GRP78-positive fibers in HIBM, which significantly higher than that in the other myopathies and normal control. (PO.001) .LIMP-1 and LC3 were up-regulated in HIBM, in which the percentage of positive fibers was 26.5+10.4% and 36.4 +8.7%, respectively. Both of them were significantly higher than that in DMD and normal control (P<0.0001) .The nuclear lamina protein , emerin, was expressed both in rimmed vacuoles and perinuclear region in HIBM, but only in perinuclear region in DMD and normal control. Other membrane proteins including SERCA, clathrin, GM-130 and porin were not overexpressed in any group.Genotype-phenotype correlation analysis of GNE gene revealed that neck muscle and quadriceps were more frequently involved in patients with heterozygous mutations than that in patients with homozygous mutation (neck muscle: 60.0% vs 25.0%, quadriceps: 70.0% vs 16.7% ). The average interval between onset and muscle biopsy in patients with heterozygous mutation was less than that in patients with homozygous mutation (5.6 yr vs 15.7yr).ConclusionWe proved apoptotic process in degenerating fibers of HIBM with different detecting methods including EM observation, in situ TUNEL method and expression of activated caspase-3. Overexpression of Caspase-12 and GRP78 in part of the muscle...