The Clinical, Biochemical, Molecular And Pharmacotherapeutic Studies Of Lipid Storage Myopathy

Metabolic myopathy is a group of skeletal muscle diseases with great clinical variation. The diagnosis needs either muscle pathology or biochemical analysis, or both. Skeletal muscle is an organ with high demand of energy. Glycogen and fatty acids are the major sources for short-term and long-term energy supply. So metabolic disorders of either glycogen or fatty acids will cause multiple systemic diseases which predominantly affects muscle system. Along with the wide use of muscle biopsy and biochemical analysis, more and more metabolic muscle disorders are diagnosed. In this study, we investigated metabolic myopathy including riboflavin responsive lipid myopathy (LSM) and neutral lipid storage disease with myopathy (NLSDM) by studies on clinical presentation, muscle pathology, biochemical analysis, molecular basis and Pharmacotherapy.LSM, defined by triglyceride accumulation in muscle fibers, is a heterogeneous group of lipid metabolic disorders exclusively or predominantly affecting skeletal muscle. Several etiological factors especially metabolic enzymes deficiency interfere with lipid catabolism in muscle cells, leading to an accumulation of light microscopic lipid droplets in muscle fibers. There are two clinical and pathological presentations, one with a lot of lipid droplets in muscle fibers and so called'lipid storage myopathy' another one with rare lipid droplets in muscle fiber, however, with acute rhabdomyolysis under stress such as exercise, fasting and infection. Along with the wide use of muscle biopsy for differential diagnosis of myopathy in China, more than200cases of LSM have been reported in Chinese literature in the past15years. The etiopathogenisis of LSM could mainly be attributed to impaired FAs oxidation in muscle fibers. Three classic LSMs were summarized genetically as follows:Primary carnitine deficiency (PCD) due to SLC22A5gene mutations; Multiple acyl coenzyme A dehydrogenase deficiency (MADD) caused by mutations in ETFA, ETFB, or ETFDH genes; Neutral lipid storage disease (NLSD) with ichthyosis (NLSDI) or with myopathy (NLSDM) due to CGI-58or PNPLA2gene mutations.In this study, we summarized the two major LSM(MADD and NLSDM) on clinical presentation, muscle pathology, biochemical analysis, molecular basis and Pharmacotherapy. These findings prompted us to gain more insight into the metabolic and genetic dysfunctions of LSM patients.Part I:Clinical, pathological and biochemical analysis of patients with lipid storage myopathyWe recruited64patients diagnosed with LSM in Neuromuscular Center of Qilu Hospital of Shandong University between1995and2010. All of these patients were subjected to muscle biopsy and pathological staining. Urine samples from22patients before riboflavin treatment and blood samples from26patients were collected for organic acid and acylcarnitine analysis, respectively.According to the clinical presentation, muscle pathology, biochemical study and responsiveness to riboflavin treatment, these64patients were divided into two different groups. Group A:61patients showed muscle weakness with neither encephalopathy nor other organs involvement, accompanied by elevated creatine kinase (CK) level in blood and muscle pain, and they were well responsive to riboflavin or low dosage of corticosteroid. Muscle pathology showed lipid droplets were prominently increased in all patients, predominantly in type I fibers. Organic acid analysis collected during crises showed an abnormal increase of a variety of dicarboxylic acids, ethylmalonic acid,2-hydroxyglutaric acid and acylcarnitine analysis revealed a combined elevation of medium (C8, C10:1, C10) and long-chain (C14:1, C14) acylcarnitines. Among64LSM patients,41were well responsive to single riboflavin (Vitamine B2) treatment (Riboflavin responsive LSM), the other20patients were not placed on this drug. Group B:There were3LSM patients showing more severe distal muscle involvement and asymmetric symptoms besides muscle weakness and exercise intolerance. Muscle pathology revealed several rimmed vacuoles. Organic acid analysis and acylcarnitine analysis didn't show any abnomalities.Part II:Study of gene mutation in patients with lipid storage myopathyIn order to find out the molecular basis in these LSM patients, we did sequence analysis on6candidate genes. Genomic DNA and total RNA were extracted from frozen muscle biopsy specimens. All coding regions and intron-exon boundaries of these genes were amplified with intron-located primers. Genomic DNA-based PCR amplifications of human ETFA, ETFB, ETFDH, MFT, ACADM and PNPLA2genes were carried out under standard conditions. The PCR products were subjected to direct bidirectional sequencing. Reverse transcription and cDNA analysis were performed in those patients with no findings on genomic sequencing.In group A, patients with riboflavin responsive LSM,57patients were confirmed with ETFDH gene mutations, which is consistent with the diagnosis of MADD. Most of the mutants were compound heterozygous located in flavin adenine dinucleotide (FAD) binding domain in ETF:QO (Electron transfer flavoprotein ubiquinone oxidoreductase) protein. There were2common mutations, c.770A>G and c.1227A>C, with frequency21and15. No mutation was found on the other4patients with riboflavin responsive LSM. In group B, the3patients with distal asymmetric LSM were proved to carry mutations on PNPLA2gene, which is consistent with the diagnosis of NLSDM.We also did haplotype analysis for linkage study in patients with two common mutations (c.770A>G and c.1227A>C) in ETFDH gene. It turned out c.770A>G doesn't belong to any haplotype block, so we couldn't calculate the recombination frequency for this mutation. And for the other mutation c.1227A>C, we didn't get any positive evidence of founder effect.For NLSDM patients with PNPLA2mutations, it revealed an extensive red staining of granulocytes on peripheral blood smear which is called Jordan anomaly, indicating there were a lot of lipid droplets in granulocytes.Part III:The effect of ETF:QO mutation on its prothetic groups FAD and CoQ10in patients with riboflavin responsive lipid storage myopathyETF:QO, encoded by the ETFDH gene, is a critical element of the pathway by which these electrons enter the Coenzyme Q10(CoQ10) pool of the main respiratory chain. In the process of fatty acid β-oxidation and amino acids catabolism in mitochondria, the electrons come into ETF:QO from electron transfer flavoprotein,(ETF) with the help of FAD, and then are transported into the respiratory chain by CoQ10. We did western blot for ETF:QO in17patients with riboflavin responsive LSM, and compare with ETF:QO protein with normal controls. It turned out ETF:QO protein expression in patients was slightly decreased. Then we purified ETF:QO protein together with its prothetic group, FAD, by immunoprecipitation, and then quantify ETF:QO protein and FAD separately to calculate the binding efficiency of these two. In patients with ETFDH gene mutations, the amount of FAD binding to the immunoprecipitated ETF:QO protein was less than that in normal controls. For muscle FAD level analysis, after normalized to total muscle weight and total protein, patients with ETFDH gene mutations had the same muscle FAD as normal controls.There are some disputations on CoQ10level in muscle of LSM patients. So it is necessary to clarify this point in our patients to see if there are any changes in CoQ10pool, the key electron receptor. CoQ10level in muscle was measured by high-performance liquid chromatography (HPLC). After normalization to muscle weight and total protein, CoQ10level in LSM patients with ETFDH gene or PNPLA2gene mutations was increased compared with normal controls (p<0.001). While after normalization to citrate synthase protein, there was no statistical difference in CoQ10level between patients with LSM and normal controls. For the determination of mRNA expression in CoQ10endogenous synthesis system and potential regulation system in LSM patients, the PDSS1, COQ2, ADCK3, PPARa, PPARβ and PPARy genes were analyzed. No up regulation was seen in CoQ10encoding genes. Increased expression of peroxisome proliferator-activated receptors (PPARs) genes was evident. PartⅣ:Therapeutic effect of β receptor agonist on lipid droplets inside fibroblast of PNPLA2mutation patientsNeutral lipid storage disease with myopathy (NLSDM) is a rare autosomal recessive disorder which is caused by mutations in PNPLA2gene (Patatin-like phospholipase domain-containing protein2, also known as CGI-58).The PNPLA2gene defect leads to a dysfunction of adipose triglyceride lipase (ATGL, also called Desnutrin) and lipid storage myopathy.It is reported that β agonists can active hormone-sensitive lipase (HSL) in fibroblast in vitro and may compensate for the disturbed or lacking function of PNPLA2. Beta adrenergic agents are drugs frequently used in the therapy of asthma and Formoterol is approved by Food and Drug Administration of the U.S. So we performed pulse-chase labeling experiment with1-pyrenedecanoic acid (PDA) and fluorescent staining experiment with Nile red and Bodipy dye for patient cells with PNPLA2gene mutations with supplementation of Formoterol in vitro. Fibrates act as synthetic ligands for PPARa which regulates lipid metabolism in peripheral tissues such as the liver and skeletal muscle, decreases circulating lipid levels, thus improving hyperlipidemia under fasting conditions. So fibroblast with Bezafibrate treatment was taken as a parallel study.The labeled fatty acid is washed out during the chase period where only unlabeled substrates are offered. Without Formoterol treatment, in cells from a control individual, the fluorescence signal disappeared on day4, whereas the clearance was less efficient in patient cells in which there was still a lot of fluorescence signal on day6. That means the turnover of lipid droplets in patient cells was much slower than that in normal control cells. With Formoterol treatment, in cells from a control individual, the fluorescence signal had disappeared on day2, so this drug makes the turnover of lipid droplets much faster in normal cells. While in cells with PNPLA2gene mutations, the remaining fluorescence signal on day6was less than the cells without drug treatment, so this drug also makes the turnover of lipid droplets faster in patient cells. But fluorescent staining experiment with Nile red and Bodipy dye didn't show changes between cells with and without Formoterol treatment, either did enzymatic quantitative study for triglyceride. It was inferred that Formoterol doesn't affect the dynamic equilibrium of lipid droplets pool in fibroblast, but just accelerate the speed of triglyceride catabolism mildly. So we should expect a significant improvement in a long term.Conclusions1. These findings suggest that the majority of Chinese patients with LSM are caused by a mild type of MADD with unique myopathy which is due to ETFDH gene mutation. And there are two common mutations in this group of patients, c.1227A>C and c.770A>G. Some of LSM patients are NLSDM due to PNPLA2gene mutation.2. The efficiency of FAD binding to ETF:QO protein was decreased caused by ETFDH gene mutations, which seems to be the pathological mechanism of MADD.3. CoQ10is not the primary factor in MADD pathogenesis. The total CoQ10pool in MADD patients was increased primarily due to an increase in mitochondrial proliferation. After normalized to mitochondria, the CoQ10level is normal in this group of LSM patients.4. Riboflavin treatment is very effective on LSM patients with ETFDH gene mutations and β agonists are very promising for LSM patients with PNPLA2gene mutations.