| BackgroundsGlycogen storage myopathy is a group of rare metabolic disorders with great clinical and pathological heterogenous.It is divided into more than ten types and characterized by vacuolar myopathy with glycogen storage in muscle biopsy.Glycogen storage type II,also known as Pompe disease,is caused by deficiency of the GAA gene which encodes the acid alfa glucosidase.Late-onset Pompe disease mainly manifested as progressive limb weakness,sometimes accompanied by respiratory insufficiency.More than 550 GAA gene mutations have been reported with different prevalent mutations in different regions and populations.Only fifty-two cases have been reported about the genetic analysis of patients from the mainland of China.Glycogen storage disease V is caused by the PYGM gene which encodes the myophosphorylase.It is characterized by exercise intolerance,myalgia,muscle contractures and myoglobinuria while sometimes there are atypical symptoms.There has been 175 PYGM gene mutations reported and p.R50X is the most common mutation.There were only two reports about the PYGM gene analysis of patients from the mainland of China.The pathogenesis of Pompe disease includes enlarged lysosomes with glycogen storage,defective autophagy,accumulation of aberrant mitochondria and dysregulation of calcium homeostasis.Autophagy is an important mechanism,but few articles have been published in China.Inflammatory reaction can be induced by the above pathogenesis,,but it has not been studied in Pompe disease.Proteomics is a high throughput technology to study the total set of proteins expressed in a given cell or biological sample at a specific development phase,physiopathologic state or environment.It is crucial for the investigation of disease pathogenesis and the searching of biomarkers for early diagnosis or new therapeutic target.The pathogenesis of Pompe disease is not very clear,so it is necessary for further investigation in the protein expression level and look for possible biomarkers.Objectives:1.To analysis the clinicopathological and genetic features of late-onset Pompe disease and McArdle disease,expanding the clinical and genetic spectrum of the two diseases.2.To preliminarily analysis the effect of autophagy and inflammation in muscle pathology of late-onset Pompe disease.3.To identify differentially regulated proteins in muscle tissues of late-onset Pompe disease and search for possible biomarkers.Methods1.Collect and analysis the clinicopathological and genetic features of 23 cases of late-onset Pompe disease and 2 cases of McArdle disease diagnosed in our department from January 2005 to January 2018.2.Using gene chips of next generation sequencing to identify the causative gene mutations and analysis the genotype-phenotype correlations.3.By using immunohistochemical technique and electron microscope,we observed the inflammatory response in 7 cases of late-onset Pompe disease and autophagy in 5 cases.4.By using iTRAQ technology and bioinformatics analysis,we identified the differentially expressed proteins in muscle tissues of 3 cases of late-onset Pompe disease compared to 3 cases of normal controls.Results1.There were 23 cases of late-onset Pompe disease with the male-to-female ratio of 1.6:1.They had disease onset at 1-67.5 years and disease course of 1.5-41 years.The main manifestation was progressive proximal limb weakness with respiratory insufficiency in 5 patients.Muscle biopsies showed unequal-sized vacuoles in muscle fibers of all patients and basophilic granules appeared in the vacuolated fibers in 82.6%of the patients.PAS staining was positive in 14 cases and negative in 9 cases,while ACP staining showed strengthened activity of acid phosphatase.There were 2 cases of McArdle disease with disease onset at 1 year and 38 years respectively.Case 1 had exercise intolerance for 17 years and case 2 had chest-back causalgia for more than a month.CK levels were 1620.8U/L and 5340U/L,respectively.Muscle biopsy showed unequal-sized subsarcolemmal vacuoles with no basophilic granules in muscle fibers.2.Genetic test revealed 29 GAA gene mutations with 7 novel ones(c.796C>A,c.1057C>T,c.1201C>A,c.1780C>T,c.1799G>C,C.2051C>A,c.2235dupG).Mutation c.2238G>C(18.18%)was the most prevalent mutation.The two patients with McArdle disease carried homozygous PYGM gene mutations c.1081G>A(p.G361R)and c.142dupA(p.T48fs),respectively,and the latter was a novel mutation.3.In 5 cases with immunohistochemical staining for LC3,LC3 was negative or weakly positive in 3 patients with severe vacuolated myofibers,positive or strongly positive in 2 patients with moderate vacuolated myofibers.4.In the muscle tissues of 7 cases with late-onset pompe disease,CD4,CD8,CD68,MHC-I was positive in 4,2,6,4 cases,respectively.SRP19 was weakly positive in 2 cases and C5B9 was negative in all cases.5.There were 168 differential proteins in patients with late-onset Pompe disease,including 131 up-regulated ones and 37 down-regulated ones.Among them,51 proteins were related to mitochondria,9 proteins were related to calium signal pathway and 5 proteins were related to autophagy.Conclusions1.Patients with late-onset Pompe disease and McArdle disease had great clinical heterogenous,but their pathological changes were characteristic.Seven novel GAA gene mutations and one novel PYGM gene mutation were identified.2.Autophagy and mild inflammatory response are involved in the muscle pathological changes of late-onset Pompe disease.3.Proteins related to mitochondria,autophagy and calium signal pathway were differentially expressed in muscle tissues of patients with late-onset Pompe disease. |
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