GNE Gene Mutations And Clinical Features Of Patients With GNE Myopathy From South China

GNE myopathy, also known as hereditary inclusion body myopathy （hIBM）, DMRV （distal myopathy with rimmed vacuoles） or Nonaka myopathy, is a rare autosomal recessive distal myopathy and caused by GNE gene （UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase gene, GenBank NM₀05476） mutation. GNE gene includes 12 exons and encodes a bifunctional enzuyme which catalyzes the biosynthesis of sialic acid. The age at onset ranges from 20 to 40 years and the patients usually become wheelchair-bound on average 10 to 12 years from the onset of symptoms. The initial symptom is weakness and atrophy of the anterior part of legs and altered gait. The disease is slowly progressive and the proximal muscles of the lower extremities, the upper extremities and the axial muscles can also be affected while the quadriceps muscles are relatively spared during the course of disease. The creatine kinase （CK） level is normal to moderately increased. Electromyogram study shows myopathic change. The presence of rimmed vacuoles is the most prominent finding on muscle biopsy. Necrotic fibers or inflammatory cellular infiltration are rarely noted or absent. Electronic microscope study shows cytoplasmic or intranuclear tubulofilamentous inclusion body. A number of cases with GNE myopathies have been reported in Japan and in the population of Milddel East Jews. Some smaller cohorts have also been reported in China. However, most of them were from Shandong province and Beijing. Study with a large sample size from south China has not yet been reported. Our study aimed to investigate GNE gene mutations spectrum in patients with typical DMRV from south China and analysis the clinical features, pathological characteristics and muscle MRI data of these patients as well as to clarify the relationship between genotype and phenotype. In order to determin the regional variation of GNE mutations in China, we also compare the patients with those previously reported in North China.Part one:GNE gene mutation analysis of GNE myopathy patients from south ChinaObjectives:Our study aimed to investigate GNE gene mutation in patients with typical DMRV from south China, comparing with the patients from north China to explore the regional differences.Methods:30 independent Han patients with typical DMRV patients in the neurology department of Huashan Hospital from 2005 to March 2014 were studied. The criteria for DMRV diagnosis included:（1） early adult onset with the weakness and atrophy of the anterior part of legs; （2） the quadriceps muscles are relative spared during the whole course of disease; （3） the creatine kinase （CK） level is normal to moderate increased; （4） Electromyogram study shows myopathic change. （5） rimmed vacuoles are not necessary for pathological presence, but inflammatory myopathies, muscular dystrophy and metabolic myopathies should be excluded. Genomic DNA was extracted from the peripheral blood leukocytes or muscle tissues and polymerase chain reaction （PCR） amplification was performed to amplify the 12 exons and their flanking intron sequences of GNE gene. All the aimed fragments were purified and analyzed by direct gene sequencing. All references to nucleotides or amino acids are based upon the genomic DNA（GRCh37.p5） sequences of GNE.Results:1.24 patients were genetically confirmed from all 30 enrolled patients, including 8 （33.3%） with homozygous mutations and 16 （66.7%） with compound heterozygous mutations. They were from Jiangxi, Zhejiang, Anhui, Jiangsu province and city of Shanghai in south China.23 of them were performed muscle biopsy and the other one was from outpatient.2.23 mutations were identified, including 18 mission mutations （78.3%）: p.C13S, p.F66V, p.I106F, p.R162C, p.D176V, p.Y186H, p.K245E, p.R246W, p.R246Q, p.K353E, p.L508S, p.H509Y, p.A524V, p. F562S, p.V572L, p.A591V, p.A638P, p.G652E; 2 nonsense mutations （8.7%）:p.Y186X, p.Q436X; 2 deletion mutations （8.7%）:p.D515fsX2（c.1543-1544delGA）, P.A600fsX43（c.1798de1G） and 1 complex double mutation （4.3%）:p.E329fsX43 （c.985delG+987-988GA>TT）. These mutations were located in the 2,3,4,6,8,9,10,11 and 12 exons. There were 10 （43.5%） reported mutations and 13 （56.5%） novel mutations:p. F66V, p.I106F, p.Y186H, p.Y186X, p.K245E, p.E329fsX43, p.K353E, p.D515fsX2, p.F562S, p.A600fsX43, p.A591V, p.A638P, p.G652E. While other parts of China reported 18 GNE gene mutations in total, and only 6 of them were same to our study.3. The three most frequent mutations were p.D176V （25.0%）, p.R246Q （12.5%）, p.K353E （10.4%）. While the three most frequent mutations in other reports of China were p.L508S （19%）, p.A631V （15%） and p.D176V （14%）. p.D176V was the same common mutation but the frequency is different from our study.Conclusions:This is the first study with large sample size of GNE myopathy patients from south China. And we first identify the deletion mutation, complex double mutation, and the mutation in the 6 and 8 exon of GNE gene in China. Compared with the north China reports, the mutations of our study have more dispersed distribution and more mutantional forms. Our mutation hotspots are also different from the north China. Besides the north China study included the familial patients, the reason of the difference between the south and the north China is perhaps related to the various geographical conditions.Part two:The clinical features and genotype-phenotype relationships of GNE myopathy patients from south ChinaObjectives:To study the clinical features, muscle MRI data, pathological findings, and the genotype-phenotype relationships of 24 genetically diagnosed GNE myopathy patients.Methods:We collected the clinical data, blood biochemical parameters and EMG results. We evaluated the adipose infiltration level of 18 muscles in Tl WI according to Mecuri criteria. The pathological materials were reviewed and the genotype-phenotype relationships were analyzed.Results:1. Male/female ratio was 13:11. Average age of onset was 33.2±8.8 （range 15-52 years）. The mean duration （from the age at symptom onset to the age at exam） of the disease was 5.04±4.4 （1-17 years）. Four （16.7%） of them had family history. The most common initial symptom was the weakness of the single or bilateral legs/drop feet （70.8%）. In physical examination, the strength of neck flexor was affected in 12 patients （50%）. The weakness of distal muscles was more severe than the proximal muscles. The quadriceps muscles were relatively spared. Their CK levels ranged from 165 to 1826 U/L （normal range:38-174 U/L） and the mean level was 477.8±380.9 U/L. The electromyogram studies showed myopathic change.2. Muscle MRI demonstrated classical involvement of the anterior compartment muscles of the lower extremities and the posterior compartment muscles of the thighs. The lateral head of the quadriceps, vastus intermedius and rectus femoris were relatively spared in the anterior compartment muscles of the thigh. And the tibialis anterior, soleus and medial head of gastrocnemius were seriously affected in the anterior compartment muscles of the leg.3.15 patients （65.2%） showed rimmed vacuoles. And necrotic fibers or inflammatory cellular infiltration were rarely noted or were absent.4. p.D176V was the most frequent mutation in this study. The onset of the disease was much later in the patients with this mutation but their strength of neck flexor was more likely to be affected. The patients with mission mutations or nonsense mutations had no differences clinical or pathological from the patients without these kinds of mutations.Conclusions:Compared with the north China reports, our patients have much later onset age. Muscle MRI is a good tool for differential diagnosis of GNE myopathy and T1WI can be used to analyze the muscle adipose infiltration level. The pathological findings are similar to the typical GNE myopathy. Rimmed vacuoles are not necessary to diagnose GNE myopathy and they are influenced by biopsy site and the durition of disease. The onset of the disease is much later in the patients with p.D176V but their strength of neck flexor is more likely to be affected.