Mutation Analysis Of The GNE Gene In 12 Chinese Patients With Hereditary Inclusion Body Myopathy

Objective:Hereditary inclusion body myopathy (HIBM) is the term currently used for vacuolar myopathy sparing quadriceps (VMSQ), and is also known as distal myopathy with rimmed vacuoles (DMRV). It was originally described by Nonaka in 1981 as an autosomal recessive distal myopthy characterized clinically by the preferential involvement of the distal muscles of the lower limbs, especially the anterior tibial muscles with sparing of the quadriceps muscles. The age at onset ranges from 15 to 40 years, with an average of 26 years. The initial symptom is usually altered gait. The disease is progressive and the patients become wheelchair-bound between 26 and 57 years of age, on average 12 years after the onset of symptoms. The serum creatine kinase (CK) levels are mildly to moderately elevated. Muscle biopsy is characterized by many rimmed vacuoles, especially in atrophic fibers. Necrotic and regenerating fibers are rarely seen. Electronic microscopic study shows cytoplasmic or intranuclear tubulofilamentous inclusion bodies in the degenerated fibers.At the beginning of this century, Eisenberg and Kayashima had identified the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) gene, which was localized on 9p12-13, as the gene responsible for HIBM and DMRV in succession. Thus HIBM and DMRV were finally proven to be the same allelic diseases with different genotypes and phenotypes according to different ethnics. After that, a lot of researchers had found different GNE gene mutations in patients with HIBM one after another.In China, Yan has first reported 9 cases of HIBM in 2003, the clinical and pathological features of whom are basically similar to those reported by Japanese. In 2005, a family with compound heterozygous mutations of the GNE gene was first reported, after that, compound heterozygotes with other GNE mutations and homozygotes were also described. But there is no more information about the GNE gene mutations of Chinese HIBM patients, so here we investigated the GNE gene of 12 patients with typical clinical and pathological features of HIBM and 6 unaffected members from 2 affected families to detect the mutations of the GNE gene in Chinese patients with HIBM and to compare the mutations between different ethnics.Material and methods:The 12 cases are all from the institute of neuromuscular disease, Qilu Hospital, Medical school of Shandong University. They were diagnosed through clinical manifestations, pathology of muscle biopsy and electronic microscopic examination. Their peripheral blood leukocytes or biopsied muscle specimens were investigated respectively. Additionally, 6 unaffected members from 2 affected families were also examined for the GNE gene mutations, their blood samples were collected by following up. Genomic DNA was extracted with phenol/chloroform procedure and polymerase chain reaction (PCR) amplification was performed to amplify all the 11 coding exons and their flanking intron sequences of GNE gene. DNA fragments were analyzed by the agarose gel electrophoresis. All the aimed fragments were purified and analyzed by direct gene sequencing, then comparison was made to detect gene mutations. Once a new mutation was found, more than 200 unrelated individuals with no history of neuromuscular diseases were tested to evaluate it.Results:1. By direct sequencing of the PCR products, a total of 5 mutations were identified in the GNE gene: a G to A substitution at np 154 (G154A) in exon 2, a A to G substitution at np 204 (A204G) in exon 2 and a A to T substitution at np 578 (A5 7 8T) in exon 3 in the epimerase domain, a T to C substitution at np 1574 (T1574C) in exon 9 and a C to T transition at np 1943 (C1943T) in exon 11 in the kinase domain, all of which were missense ones and resulted in the amino acid substitutions glutamic acid to lysine at codon 35 (E35K), isoleucine to methionine at codon 51 (I51M), aspartic acid to valine at codon 176 (D176V), leucine to serine at codon 508 (L508S) and alanine to valine at codon 631 (A631V) respectively. A5 78T and C1943T had already been found in Japanese HIBM patients, T1574C had already been found in Chinese patients, while G154A and A204G had not been reported yet. They were not found in more than 200 unrelated individuals with no history of neuromuscular diseasese, suggesting that they were novel pathogenic mutations.2. 8 of the 12 patients were found to carry mutations in the GNE gene, all of whom were women. 5 were compound heterozygotes: 3 had T1574C and C1943T mutations, including two sisters from a family and the other 2 siblings bore compound heterozygous mutations of A204G and T1574C. 3 were simple heterozygotes of the gene with mutations G154A, A578T and T1574C respectively and the other 4 were found to have no mutations of the gene. None of the patients were homozygotes.3. Each of the 6 unaffected persons of the 2 affected families carried only simple heterozygous mutation of the GNE gene. The two heterozygous mutations of the patients came from the paternal and maternal chromosomes respectively.Conclusions:1. A high frequency (66.7%) of the GNE gene mutation is found in Chinese patients with HIBM, although lower than that of the Japanese patients (91% and 79.4%).2. Of the Chinese HIBM patients we examined, some are compound heterozygotes, another are simple heterozygotes and the others are found to have no mutations in the GNE gene and no homozygotes are found, which is different from that almost all of the patients in Middle Eastern Jews had a single homozygous missense mutation T2186C (M712T) in exon 12 of the gene, while about 30 different mutations in either homozygous or compound heterozygous state were identified in.the Japanese DMRV patients. There are already homozygotes reported in China.3. It is accordant with the foreign patients that most of the mutations of the GNE gene are missense ones and no homozygous null mutations are found. The mutations can be in either the epimerase or the kinase domain of the protein.4. Of the 5 pathogenic mutations, A5 78T and C1943T have already been reported in the Japanese patients, suggesting that there is some likeness between the patients of the two countries; G154A, A204G and T1574C are not found in the foreign patients. G154A and A204G are two novel mutations that are first described in this study. T1574C has already been reported in China, 6 of the 8 positive patients are found to carry it, suggesting that it may be a more common mutation in Chinese HIBM patients.5. There are no clinical or pathological differences between compound heterozygotes, simple heterozygotes and the patients without mutations in the GNE gene.