GNE Gene Mutation Analysis In A Chinese Family Of Distal Myopathy With Rimmed Vacuoles

Objective:Distal myopathy with rimmed vacuoles (DMRV) is an autosomal recessive inherited neuromuscular disorder which was first reported by Nonaka in 1981. The onset of the disease ranged from 15 to 40 years, averaging 26 years. The initial symptoms are gait disturbances, clinically characterized by early involvement of distal muscles of low limbs, especially anterior tibial muscles. Though Proximal muscles are involved in late stage, quadriceps is constantly spare. The disease was progressive and patients become non-ambulant averaging 12 years after the onset of the disease. The serum creatine kinase (CK) levels were mildly to moderately elevated.The characteristic pathological findings is rimmed vacuole foundation in atrophic muscle fibers with little evidence of necrotic or regenerative progress. Electronic microscropic study shows cytoplasmic inclusion bodies and intranuclear filamentous inclusions formation in the degenerated fiber with rimmed vacuoles. Yan has reported 9 cases of DMRV occurred in China. The clinical and pathological features is basically similar to those reported by Japanese.In 2002, Kayashima found UDP-N-acetylglucosamine2-epimerase/acetylmannosamine kinase (GNE) gene mutation in DMRV, which waslocalized on 9P12-13 and encoded a bifunctional enzyme. Nishino identified that hereditary inclusion body myopathy (HIBM) , originally described in 1983 as "rimmed vacuole myopathy" sparing the quadriceps , is an autosomal recessive disorder very similar to DMRV not only clinically but also pathologically. Both diseases were mapped to the same gene. Thus HIBM and DMRV finally proved to be the same allelic diseases. Although DMRV and HIBM are caused by mutations in the GNE gene , the genotype are not alike according to different ethnics. A single homozygous missense mutation in exon 12 of the GNE gene is responsible for all HIBM patients in Middle Eastern Jews. In contrast, about 20 different GNE mutations in either homozygous or compound heterozygous states were identified in Japanese patients. According the literature, Japanese DMRV patients seem to develop severer consequence compared with those in Middle Eastern Jew, Up to now, no mutations in GNE about Chinese DMRV were reported, therefore, the analysis of mutation and genotype-phenotype correlation in our country is required. Here we investigated the GNE gene of two siblings with DMRV from a Chinese family and explore the genotype-phenotype correlation of GNE gene mutation in different ethnics. Material and methods:The two cases are from the department of neurology of Qilu hospital, Shandong university. They were diagnosed through clinical manifestation, pathology of muscle biopsy, and electron-microscopic examination. Genomic DNA was extracted from peripheral blood of the affected family that were followed up, with phenol/chloroform procedure. Exon 1 to 13 of GNE gene were amplified by polymerase chain reaction (PCR).DNA fragment was analyzed by the agrose gel electrophoresis . We sequenced all the exons and their flanks for detecting gene mutation. Genotype-phenotype correlations were analyzed with summarized clinical and pathologicalfeatures of the two DMRV patients.Results:1. Pedigree of the family: The affeted family had 10 persons and the two siblings are both DMRV diagnosed through clinical manifestation, pathology of muscle biopsy, and electron-microscopic checking.2.Cliniacal manifestation features:They were first noticed to have walking disturbance in late adolescence. And also complained of difficulty climbing stairs and running, and frequent falls. Distal muscle weakeness and atrophy of the lower extremities was predominant in early stage. Proximal and trunk muscles were involved in the advanced stage, especially anterior tibial and iliopsoas muscles, sparing quadriceps femoris muscle. The serum creatine kinase (CK) levels were mildly elevated.This disease was progressive. The two patients became non-ambulant about 10 years after the onset of the disease.3. Pathological features: Rimmed vacuoles formation and muscle fiber atrophy without marked necrotic and regenerating process was the most prominent alteration under light microscope. No inflammatory cells, no glycogen or lipid storage, and no typical fiber type grouping were found. 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 stricking findings in the two siblings.4. Gene mutations:Direct sequencing of GNE revealed that both patients were compound heterozygous for a T to C substitution at np 1574 in exon 9, converting leucme to serine at codon 508 (L508S), and a C to T transition at np 1943 in exon 11, converting alanine to valine at codon 631 (A631V). A631V mutation was found in her father, while her mother ,sister andbrother all carried the L508S mutation. Whereas the L508S transition in exon 9 is a new mutation, the A631V transition corresponds to that described in both Tomimitsu and Nishino. In our patients the two mutations identified are both in kinase domain of the protein, possibly leading to an impairment of enzyme catalytic activity. Conclusion:1.Chinese DMRV patients seem to develop similar clinical and pathological features compared with those in Japanese. The initial symptoms are gait disturbances, clinically characterized by early involvement of distal muscles of low limbs, especially anterior tibial muscles. Though Proximal muscles are involved in late stage, quadriceps is constantly spare. The characteristic pathological findings is rimmed vacuole foundation in atrophic muscle fibers and electronic microscropic study shows cytoplasmic inclusion bodies and intranuclear filamentous inclusions formation in the degenerated fiber with rimmed vacuoles.2. This is the first report on mutations of GNE in distal myopathy with rimmed vacuoles in China, the two compound heterozygous are both in kinase domain of the protein.3. In this Chinese family with DMRV, two patients carried a compound heterozygous mutation in the GNE gene. One of their mutations (L508S) was found no report before, whereas the other mutation (A631V) has been reported in Japanese DMRV, indicating a strong founder effect in Chinese and Japanese DMRV pedigree.