A Comparative study on riboflavin responsive multiple acyl-CoA dehydrogenation deficiency due to variants in FLAD1 and ETFDH gene

A Comparative study on riboflavin responsive multiple acyl-CoA dehydrogenation deficiency due to variants in FLAD1 and ETFDH gene | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article A Comparative study on riboflavin responsive multiple acyl-CoA dehydrogenation deficiency due to variants in FLAD1 and ETFDH gene Chuanzhu Yan, Bing Wen, Runqi Tang, Shuyao Tang, Yuan Sun, Jingwen Xu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2314639/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Lipid storage myopathy (LSM) is a heterogeneous group of lipid metabolism disorders predominantly affecting skeletal muscle by triglyceride accumulation in muscle fibers. Riboflavin therapy has been shown to ameliorate symptoms in some LSM patients who are essentially concerned with multiple acyl-CoA dehydrogenation deficiency (MADD). It is proved that riboflavin responsive LSM caused by MADD is mainly due to ETFDH gene variant ( ETFDH- RRMADD). We described here a case with riboflavin responsive LSM and MADD resulting from FLAD1 gene variants (c.1588C > T p.R530C and c.1589G > C p.R530P, FLAD1- RRMADD). And we compared our patient together with 9 FLAD1- RRMADD cases from literature to 106 ETFDH- RRMADD cases in our neuromuscular center on clinical history, laboratory investigations and pathological features. Furthermore, the transcriptomics study on FLAD1- RRMADD and ETFDH- RRMADD were carried out. On muscle pathology, both FLAD1 -RRMADD and ETFDH -RRMADD were proved with lipid storage myopathy in which atypical ragged red fibers were more frequent in ETFDH -RRMADD, while fibers with faint COX staining were more common in FLAD1 -RRMADD. Molecular study revealed that the expression of GDF15 gene in muscle and GDF15 protein in both serum and muscle was significantly increased in FLAD1- RRMADD and ETFDH -RRMADD groups. Our data revealed that FLAD1- RRMADD (p.R530) has similar clinical, biochemical, and fatty acid metabolism changes to ETFDH -RRMADD except for muscle pathological features. Biological sciences/Genetics/Clinical genetics/Disease genetics Health sciences/Diseases/Neurological disorders/Neuromuscular disease lipid storage myopathy riboflavin multiple acyl-CoA dehydrogenation deficiency FLAD1 gene ETFDH gene GDF15 gene Figures Figure 1 Figure 2 Figure 3 Introduction Lipid storage myopathy (LSM) is a heterogeneous group of lipid metabolism disorders characterized by impaired oxidation of fatty acids (FAs) predominantly affecting skeletal muscle by intramuscular triglyceride accumulation. Several common pathogenic factors are as follows[ 1 , 2 ]: multiple acyl-coenzyme A dehydrogenation deficiency (MADD), neutral lipid storage disease with myopathy (NLSDM), neutral lipid storage disease with ichthyosis (NLSDI) and primary carnitine deficiency (PCD). Rare causes are reported such as long-chain L-3-hydroxyacyl-coenzyme A dehydrogenase deficiency (L-CHADD) [ 3 ], short-chain acyl-coenzyme A dehydrogenase deficiency (SCADD)[ 4 ] and Very long-chain acyl-CoA dehydrogenase deficiency (VLCADD)[ 5 , 6 ]. Among these, only MADD-LSM have been reported to be well responsive to riboflavin[ 7 , 8 ]. MADD (MIM 231680; also known as glutaric acidemia type II, GAII) has a wide range of severity varying from neonatal lethal form to late onset milder form presenting with only myopathy. The majority of the late-onset patients benefit from riboflavin therapy[ 9 ]. The genetic background of late-onset MADD is heterogeneous and most of these patients carry variants in electron transfer flavoprotein dehydrogenase gene ( ETFDH ), while a minority have variants in the genes encoding the alpha ( ETFA ) and beta ( ETFB ) subunits of the electron transfer flavoprotein[ 10 ]. It is reported that riboflavin transporter genes such as SLC52A1 , SLC52A2 and SLC52A3 [ 11 – 15 ], mitochondrial FAD transporter gene SLC25A32 [ 16 ] as well as flavin adenine dinucleotide synthase (FADS) gene genes FLAD1 [ 17 ] are related to riboflavin responsive MADD like phenotype[ 18 ]. Recently, more cases with FLAD1 variants were reported as a cause of MADD, which presents heterogeneous response to riboflavin treatment depending on different genotype[ 17 ]. We described here a patient with riboflavin responsive LSM and MADD resulting from FLAD1 gene variant ( FLAD1 -RRMADD) and carried out studies on gene expression between FLAD1- RRMADD and ETFDH- RRMADD to investigate why these two genes with different functions cause such similar phenotype. We reviewed literature and summarized 9 cases with riboflavin responsive FLAD1 gene variants, followed by comparing these 10 cases with 106 ETFDH- RRMADD cases in our neuromuscular center on clinical history, laboratory investigations and muscle pathology. Methods Patient Between 1995 and 2019 at Research Institute of Neuromuscular and Neurodegenerative Diseases, Qilu Hospital, Shandong University, a total of 106 patients with riboflavin responsive late-onset MADD were identified with ETFDH gene variants ( ETFDH- RRMADD), only one case with riboflavin responsive late-onset MADD was proved with FLAD1 gene variants ( FLAD1 -RRMADD)[ 9 ]. This patient with FLAD1 gene variants has been followed-up regularly for over 7 years after he was diagnosed in 2015. This retrospective study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the medical Ethics Committee of Qilu Hospital (number: KYLL-202208-024). Informed consent for diagnostic muscle biopsy, research and publication was obtained from all individual participants included in the study (106 ETFDH- RRMADD and one FLAD1 -RRMADD). Muscle pathology Open muscle biopsy in left biceps brachii was carried out under local anaesthesia. Muscle specimens were frozen in isopentane that was precooled in liquid nitrogen and stored at -80℃. For histological examination, serial frozen sections (8 mm) were stained with hematoxylin-eosin (H&E), Oil red O (ORO), succinate dehydrogenase (SDH), modified Gomori trichrome (mGT), nicotinamide adenine dinucleotide-tetrazolium reductase (NADH-TR), cytochrome c oxidase (COX) and periodic acid Schiff (PAS). For immunohistochemistry studies, serial frozen sections (5 mm) were incubated with dystrophin, dysferlin, α-sarcoglycan, β-sarcoglycan, γ-sarcoglycan, δ-sarcoglycan, and caveolin-3 antibodies. Transcriptomics Total amounts and integrity of RNA from muscle before riboflavin treatment were assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). Total RNA was used as input material for the RNA sample preparations. After the construction of the library, the library was initially quantified by Qubit2.0 Fluorometer, then diluted to 1.5ng/ul, and the insert size of the library is detected by Agilent 2100 bioanalyzer. After insert size meets the expectation, qRT-PCR is used to accurately quantify the effective concentration of the library to ensure the quality of the library. The different libraries are pooling according to the effective concentration and the target amount of data off the machine, then being sequenced by the Illumina NovaSeq 6000. The end reading of 150bp pairing is generated. The basic principle of sequencing is to synthesize and sequence at the same time (Sequencing by Synthesis). FeatureCounts (v1.5.0-p3) was used to count the reads numbers mapped to each gene. And then FPKM (Fragments Per Kilobase Million) of each gene was calculated based on the length of the gene and reads count mapped to this gene. Based on the gene expression level, the differentially expression genes (DEGs) between samples can be identified. With DEGs, we perform Gene Ontology (GO) classification, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway classification and functional enrichment. GDF15 level detection in serum The analysis of GDF15 level in serum was carried out according to the instructions of a human GDF15 ELISA Kit (ab155432, Abcam Plc, United Kingdom) Literature review and comparation study We compared FLAD1- RRMADD (our case together with cases review from literature) and ETFDH- RRMADD in our neuromuscular center (106 cases)[ 9 ]. Statistics 1.Comparative study Statistical analysis was conducted with SPSS v.12.0 software package for Windows (SPSS Inc.), applying the paired Student's t test and two-sided Student's t test. The categorical variables were studied using Chi-square analysis. The numerical variables were studied using t-test. Two-sided p values were computed, and p < 0.05 was considered statistically significant. 2.Transcriptomics study For DESeq2 with biological replicates: Differential expression analysis of two conditions/groups (two biological replicates per condition) was performed using the DESeq2 R package (1.20.0). DESeq2 provide statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The resulting P-values were adjusted using the Benjamini and Hochberg’s approach for controlling the false discovery rate. padj =1 were set as the threshold for significantly differential expression. For edgeR without biological replicates: Prior to differential gene expression analysis, for each sequenced library, the read counts were adjusted by edgeR program package through one scaling normalized factor. Differential expression analysis of two conditions was performed using the edgeR R package (3.22.5). The P values were adjusted using the Benjamini & Hochberg method. padj =1 were set as the threshold for significantly differential expression. Results Clinical features and follow up The patient was 56-year-old male. His developmental milestones were normal. During childhood he was able to run as fast as his peers. The limb strength was normal before he was 51-year-old, when he had progressive muscle weakness on lower extremities. His upper extremities were involved one month ago. When consulted, he complained with difficulty in standing up from squatting position and lifting arms over his shoulder, as well as tiredness after walking for 100 meters and muscle tenderness. He didn’t describe any difficulty with drinking, chewing or swallowing. There was a past history of cigarettes smoking. Alcohol and substance use was denied. There was no family history of muscle disease and the pedigree was shown in Fig. 1A. Neurologic examination revealed that he was awake, alert and interactive. Vision and speech normal. Pupils are equal and reactive. Extraocular movement intact. Facial strength normal. No weakness on eye or mouth closure. Tongue movements normal. Motor strength using the medical research council (MRC) scale: Neck flexion and extension 5/5. deltoids 3/5, biceps 4/5(left) and 4+/5(right), triceps 5-/5(left) and 5/5(right), wrist extension and flexion 5/5, finger extension and flexion 5/5, hip flexion 4/5, knee and ankle extension or dorsiflexion 5/5. Muscle tone normal. No muscle atrophy. Sensory examination intact to pinprick, vibration, and position. Reflexes 2 + symmetrical. He had continuously elevated creatine kinase(CK) fluctuating between 383 to 928 U/L (38–174 U/L). Needle EMG showed myogenic pattern on deltoids and quadriceps; Sensory and motor nerves were normal; F wave, H reflex and repeating nerve stimulation were normal; No myotonic discharge. The ultrasound of heart, liver, gallbladder, pancreas, spleen and kidney as well as electrocardiogram was normal. The second generation sequencing using blood sample was carried out for gene analysis, and it revealed compound heterozygous variants in FLAD1 (c.1588C > T p.R530C reported previously[ 17 ]; c.1589G > C p.R530P ; Fig. 1B). Although DNA sample from the patient’s parents who passed away at an advanced age was not available, sequence analysis on Bam picture proved that variants c.1588C > T and c.1589G > C were from two isolated homologous chromosomes(Fig. 1C). Western blot against FADS ( FLAD1 gene) in muscle sample before riboflavin treatment was significantly reduced in this patient (Supplemental material 1). His muscle strength was normal after 2 months treatment with riboflavin 80mg/day. Then he was placed on a reduction dose of riboflavin 30 mg/day in the following 7 years. During follow-up, his CK level, needle EMG on deltoids, the ultrasound examination of heart and electrocardiogram were normal. The riboflavin level in serum was 10.8 ng/ml (2.3–14.6 ng/ml) after treatment. Acylcarnitine profile from dried blood spots revealed moderate elevations of C4-, C5-, C6-, C8-, C10-, C10:1-, C12-, C14:1-, C14:2- and C18:2-acylcarnitines. Urine organic acid analysis showed mild elevation of ethylmalonic acid. Muscle MRI showed muscle atrophy and fatty degeneration on the posterior thigh group on T2WI-FSE (Fast spin echo) after 5 years of riboflavin treatment, moreover, the fat infiltration signal was suppressed on T2WI-FSE-FS (Fat suppression) (Fig. 1D). Muscle pathology Light-microscopic assessment of histochemical stains showed a marked variation in fiber size due to a large number of small atrophic fibers. Fibers with centrally placed nuclei accouted for about 20%. A few highly atrophic fibers formed nuclear clumps (Black arrow in Fig. 2A). There were a lot of fibers with small round vacules (Stars in Fig. 2A) which were proved as type I fiber in ATPase staining (Fig. 2G and 2H). There were a few necrotic and regenerating fibers and atypical ragged red fibers(RRFs, Stars in Fig. 2B), as well as numerous COX-negative fibers(Stars in Fig. 2C). The ORO stain showed that lipid droplets were prominently increased predominantly in type I fibers (Fig. 2D). Diffuse SDH enzyme deficiency was noticed compared to normal control(Fig. 2E and 2F, separately). There was no group atrophy and the atrophic fibers are mainly type I (Fig. 2G and 2H). The immunohistochemistry studies did not show any abnormality. Transcriptomic study To elucidate the underlying mechanisms of RRMADD, the whole transcriptome sequencing together with integrated bioinformatics analysis were conducted in this patient with FLAD1- RRMADD, three patients with ETFDH- RRMADD and three normal controls. Venn diagram was used to display expressed gene between three groups shown in Fig. 3A. Summary of DEGs was shown in Fig. 3B. The results showed a total of 1610 DEGs between FLAD1 -RRMADD and control group, among these 709 up-regulated and 901 down-regulated DEGs were identified, involving numerous signaling pathways (Supplemental material 2 − 1); for that between ETFDH -RRMADD and control group, a total of 1143 DEGs, 623 up-regulated and 520 down-regulated DEGs were identified (Supplemental material 2–2); for FLAD1 -RRMADD and ETFDH -RRMADD group, a total of 1019 DEGs, 354 up-regulated and 665 down-regulated DEGs were identified (Supplemental material 2–3). Volcanic maps showed that the expression of GDF 15 mRNA was overwhelmingly significant up-regulated in both FLAD1 -RRMADD vs control and ETFDH -RRMADD vs control group (Fig. 3C and 3D). We next detected the GDF15 protein level in serum in FLAD1 -RRMADD after riboflavin treatment (N = 1) and ETFDH -RRMADD (before and after riboflavin treatment, N = 5). The GDF15 level in serum was an obvious increase in ETFDH -RRMADD group before and after riboflavin treatment comparing to normal controls (N = 10, p < 0.001, Fig. 3E). For FLAD1 -RRMADD, the GDF15 level in the only patient after riboflavin treatment was significantly higher than the average of 10 normal controls (862.75 vs 342.68). The protein expression of GDF15 in muscle samples before riboflavin treatment was proved significantly increased in both FLAD1 -RRMADD and two ETFDH -RRMADD patients (Supplemental material 1). Volcanic maps also showed that the expression of TRIB3 and KLHDC7B mRNA was significant up-regulated in both FLAD1 -RRMADD vs control and ETFDH -RRMADD vs control group (Fig. 3C and 3D). Nevertheless, the protein study for those two genes failed to support the results that were found in mRNA study. GO classification for the co-expressed genes of FLAD1 -RRMADD vs control and ETFDH -RRMADD vs control showed that the main differences were fatty acid metabolism process (Fig. 3F). KEGG pathway classification analysis for the co-expressed genes of FLAD1 -RRMADD vs control and ETFDH -RRMADD vs control showed that the main differences were biosynthesis of amino acids and fatty acid metabolism (Fig. 3G). Comparison between FLAD1 -RRMADD and ETFDH -RRMADD We reviewed literature and listed a summary of 18 cases with MADD/GAII caused by FLAD1 variants including the present case (Supplemental material 3), in which 10 patients were proved with full or partial riboflavin responsiveness(Full riboflavin responsiveness: After riboflavin treatment, the symptoms including muscle weakness disappear and the patient returns to a normal life. Partial riboflavin responsiveness: After riboflavin treatment, the symptoms are partially improved, the patient' condition is stable but not normal) [ 9 , 17 , 19 – 23 ]. The comparison between 10 FLAD1 -RRMADD (9 cases from literature and the present case) and 106 ETFDH -RRMADD (in our neuromuscular center) was shown in Table 1 . Table 1 The comparison for clinical, biochemical and pathological characteristics between FLAD1 -RRMADD and ETFDH -RRMADD MADD/GAII patients FLAD1 (10 cases) ETFDH (106cases) p value Sex(M:F) 5:5 61:45 Onset age, mean (range) 14.73y(2m-51y) 29.2y(5y-73y) Symmetric limb girdle weakness 8/10 106/106 Craniofacial and neck muscle weakness 7/10 89/106 0.26 Vomiting 4/10 23/106 0.19 Myogenic pattern in EMG 4/5 28/53 0.24 CK(U/L), mean(range) 2044.2 (380–6612) 1504.8 (102-11022) Muscle pathology Lipid storage 7/7 106/106 Atypical RRF 1/7 84/106 < 0.001 Decreased SDH activity 2/7 69/106 0.52 Faint COX staining 4/7 0/106 < 0.001 Homozygous mutation 7/10 8/106 CK, serum creatine kinase (normal value: 26–178 mmol/l); The onset age of FLAD1 -RRMADD ranges from birth to 51 years, in which 6 out of 10 cases presented in infancy, in contrast, cases with ETFDH -RRMADD tend to be late-onset. On muscle pathology, 100% of FLAD1 -RRMADD or ETFDH -RRMADD were proved with lipid storage myopathy. The atypical RRFs were more frequent in ETFDH -RRMADD, while the fibers with faint COX staining were more common in FLAD1 -RRMADD (p < 0.001, respectively). On muscle weakness symptom, accompanying symptoms such as vomiting, electromyography, serum CK level, and other muscle pathology features such as lipid storage or decreased SDH activity, it was similar between FLAD1 -RRMADD and ETFDH -RRMADD. Discussion We described here a case with compound heterozygous FLAD1 -RRMADD (c.1588C > T p.R530C and c.1589G > C p.R530P in the FADS domain), and followed-up this patient for 7 years. The patient with FLAD1 variants was characterized by late-onset progressive proximal muscle weakness, lipid storge myopathy and dramatic good responsiveness to riboflavin treatment, which were extremely similar to ETFDH -RRMADD. After this patient was clinically improvement, it was proved that his plasma acylcarnitine was still abnormal featured by elevated short, medium and long chain acylcarnitine, as well as slightly increased ethylmalonic acid in urine organic acid. After riboflavin treatment for 5 years, although his muscle strength was normal, it was proved that this patient still had obvious muscle atrophy and fatty degeneration in posterior thigh group on muscle MRI, as reported previously in medial calf muscles[ 21 ]. To date, there were totally 17 MADD/GAII cases from 9 countries caused by FLAD1 variants according to the publications until April, 2022[ 9 , 17 , 19 – 26 ]. Among those cases, 9 patients were proved with full or partial riboflavin responsiveness. Then, in order to learn more information on FLAD1 -RRMADD, after comparing 10 FLAD1 -RRMADD (9 cases from literature and one case in our neuromuscular center) and 106 ETFDH -RRMADD (in our neuromuscular center), some new understanding on this disease was added. The FLAD1 -RRMADD could be infancy onset or late-onset. Except for lipid storage myopathy, the fibers with faint COX staining were more common in FLAD1 -RRMADD on muscle pathology. The proportion of consanguineous homozygotes was higher in FLAD1 -RRMADD. On the other clinical, biochemical or pathological features, it was similar between FLAD1 -RRMADD and ETFDH -RRMADD. It is hard to differentiate the two types of RRMADD being independent of genetic testing. Among 17 MADD/GAII cases caused by FLAD1 variants in publication, 9 cases were full or partially responsive to riboflavin treatment. Meanwhile riboflavin regimen was not administrated in 4 patients and was discontinued due to side effects in one patient, for this reason we are not sure whether those 5 patients could be cured by riboflavin treatment[ 17 , 24 ]. Only three patients with nonsense or frameshift variants (homozygous p.Arg249∗; p. Arg109Ala fs*3 and p.Ser167Pro fs*20; p.Glu266Val fs*3 and splice site variant) were reported as poor responsiveness to riboflavin treatment[ 17 , 25 , 26 ]. Whether to be responsive to riboflavin may be dependent on the genotype of FLAD1 variants. It is proved that the phenotype with frameshift variants seems more severe than that harboring single amino acid changes in the FADS domain[ 17 ], that may account for the different responsiveness to riboflavin in different patients. The fact that our case carried two heterozygous amino acid changes in FADS domain and was well responsive to single riboflavin treatment supported the theory above. Transcriptomics study and GDF15 protein detection showed that the expression of GDF15 mRNA in muscle and protein in both serum and muscle was significant up-regulated in FLAD1 -RRMADD and ETFDH -RRMADD groups. GDF15 is a stress response cytokine that regulates energy metabolism. For now, we are unclear whether the increase of GDF15 is a cause or a result in RRMADD patients. The expression of GDF15 receptor, GFRAL/RET was not upregulated in this study suggesting that the increase of GDF15 might be a result of abnormal energy metabolism. On the other hand, it was reported that GDF15 could activate AMPK pathway in the absence of the GDF15 receptor GFRAL in skeletal muscle[ 27 ], and GDF15 is produced by skeletal muscle but targets adipose tissue to promote lipolysis[ 28 ], both of which may support that the increase of GDF15 is a cause of abnormal energy metabolism. Hence, future studies of the effects of GDF15 in lipid storage myopathy are highly needed. Gene enrichment analysis revealed that fatty acid metabolism was upregulated in both FLAD1 -RRMADD and ETFDH -RRMADD, which may result from negative feedback regulation involving in pathological process of lipid storage in skeletal muscle. Transcriptomics study also showed that the expression of TRIB3 and KLHDC7B mRNA was significant up-regulated in both FLAD1 -RRMADD and ETFDH -RRMADD except for GDF15 . We have done protein analysis and searched these genes function in NCBI database or Pubmed literature, also done analysis of protein protein interaction (PPI) network using STRING protein interaction database or Cytoscape software. After all these studies, only GDF15 is the most likely key gene that may involve in the pathogenesis of both FLAD1 -RRMADD and ETFDH -RRMADD. And an ongoing work was performing in our lab on GDF15 with a hybrid mice model of gdf15 −/− and etfdh −/− . We look forward to finding out something new. Overall, we described a case with FLAD1 -RRMADD who has similar clinical, biochemical, and fatty acid metabolism changes to ETFDH -RRMADD, as well as the up-regulated expression of GDF15 gene in muscle and GDF15 protein in serum. A comparative study on FLAD1- RRMADD and ETFDH- RRMADD revealed that COX deficiency fibers might be a pathological marker for differentiating FLAD1 -RRMADD from ETFDH -RRMADD. Declarations ACKNOWLEDGEMENTS The authors are grateful to the patient and his families for their participation. CONFLICTOFINTEREST The authors report no conflict of interest related to this article. DATA AVAILABILITY STATEMENT The data that support the findings of this study are available from the corresponding author upon reasonable request. ETHICAL APPROVAL The study was approved by the Ethics Committee of Qilu Hospital, Shandong University (number: KYLL-202208-024). Informed consent was obtained from all patients for being included in the study. FUNDING This work was supported by the National Natural Science Foundation of China (No.82071412); the Youth Program of National Natural Science Foundation of China (81701058，81901278); National Key Research and Development Program of China (2021YFC2700904); Qingdao Technology Program for Health and Welfare (20–3-4–42-nsh); the Taishan Scholars Program of Shandong Province. ORCID ID Bing Wen 0000-0001-8141-6662 Tan Wang 0000-0002-1061-161X Chuanzhu Yan 0000-0002-2191-5184 References Bruno C and Dimauro S Lipid storage myopathies. Curr Opin Neurol. 2008;21:601–6. Liang WC and Nishino I Lipid storage myopathy. Curr Neurol Neurosci Rep. 2011;11:97–103. Fryburg JS, Pelegano JP, Bennett MJ, and Bebin EM Long-chain 3-hydroxyacyl-coenzyme A dehydrogenase (L-CHAD) deficiency in a patient with the Bannayan-Riley-Ruvalcaba syndrome. Am J Med Genet. 1994;52:97–102. Tein I, Elpeleg O, Ben-Zeev B, Korman SH, Lossos A, Lev D, et al. 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Garcia-Villoria J, De Azua B, Tort F, Mosegaard S, Ugarteburu O, Texido L, et al. FLAD1, encoding FAD synthase, is mutated in a patient with myopathy, scoliosis and cataracts. Clin Genet. 2018;94:592–3. Aguilar-Recarte D, Barroso E, Guma A, Pizarro-Delgado J, Pena L, Ruart M, et al. GDF15 mediates the metabolic effects of PPARbeta/delta by activating AMPK. Cell Rep. 2021;36:109501. Laurens C, Parmar A, Murphy E, Carper D, Lair B, Maes P, et al. Growth and differentiation factor 15 is secreted by skeletal muscle during exercise and promotes lipolysis in humans. JCI Insight. 2020;5. Additional Declarations There is no duality of interest Supplementary Files Supplementalmaterial1.docx Supplementalmaterial21ThedifferentiallyexpressiongenesbetweenFLAD1RRMADDandcontrolgroup.xlsx Supplementalmaterial22ThedifferentiallyexpressiongenesbetweenETFDHRRMADDandcontrolgroup.xlsx Supplementalmaterial23ThedifferentiallyexpressiongenesbetweenFLAD1RRMADDandETFDHRRMADDgroup.xlsx Supplementalmaterial3XXXXXXXXXXXXXXX.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2314639","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":159884500,"identity":"8678212c-ea0d-499a-9ed0-34b71be96c80","order_by":0,"name":"Chuanzhu Yan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvElEQVRIiWNgGAWjYNCCCgbGBiAlQYKWMyRrYWwjRQt//xnDz4Xz6mQ3HGA+eJuHwS6PoBaJA2eMpWduO2y84QBbsjUPQ3IxQS0GjD0G0rzbDiRuOMBjJs3DcCCxgaAWZh7j37xz6oBa+L8RqYUNaDhvAzPIFjbitEicYSuz5jl22HjmYTZjyzkGyYS18Pcf3nybp6ZOtu9488MbbyrsCGthYOAwgNDMYHcSVg8E7A+IUjYKRsEoGAUjGAAALOM34oJZQcEAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-2191-5184","institution":"Qilu Hospital, Shandong University","correspondingAuthor":true,"prefix":"","firstName":"Chuanzhu","middleName":"","lastName":"Yan","suffix":""},{"id":159884501,"identity":"ecd490b0-97f3-4ab0-92e9-a7ffc055c1bb","order_by":1,"name":"Bing Wen","email":"","orcid":"","institution":"Qilu Hospital, Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Bing","middleName":"","lastName":"Wen","suffix":""},{"id":159884502,"identity":"91f8db38-9cff-4612-b1da-e5c4d36b1820","order_by":2,"name":"Runqi 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University","correspondingAuthor":false,"prefix":"","firstName":"Tan","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2022-11-26 08:20:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2314639/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2314639/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":30445243,"identity":"8ae22764-0704-41e3-ad8c-81ced9882561","added_by":"auto","created_at":"2022-12-16 18:26:51","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1123687,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-2314639/v1/44b596361ea47adb6e205c38.png"},{"id":30444589,"identity":"7b0a1176-cfab-48e6-9cbd-f9fc3a36b00d","added_by":"auto","created_at":"2022-12-16 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18:10:52","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":59832,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementalmaterial3XXXXXXXXXXXXXXX.docx","url":"https://assets-eu.researchsquare.com/files/rs-2314639/v1/7e618abb8287111c26fed5cf.docx"}],"financialInterests":"There is no duality of interest","formattedTitle":"A Comparative study on riboflavin responsive multiple acyl-CoA dehydrogenation deficiency due to variants in FLAD1 and ETFDH gene","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLipid storage myopathy (LSM) is a heterogeneous group of lipid metabolism disorders characterized by impaired oxidation of fatty acids (FAs) predominantly affecting skeletal muscle by intramuscular triglyceride accumulation. Several common pathogenic factors are as follows[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]: multiple acyl-coenzyme A dehydrogenation deficiency (MADD), neutral lipid storage disease with myopathy (NLSDM), neutral lipid storage disease with ichthyosis (NLSDI) and primary carnitine deficiency (PCD). Rare causes are reported such as long-chain L-3-hydroxyacyl-coenzyme A dehydrogenase deficiency (L-CHADD) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], short-chain acyl-coenzyme A dehydrogenase deficiency (SCADD)[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] and Very long-chain acyl-CoA dehydrogenase deficiency (VLCADD)[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Among these, only MADD-LSM have been reported to be well responsive to riboflavin[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMADD (MIM 231680; also known as glutaric acidemia type II, GAII) has a wide range of severity varying from neonatal lethal form to late onset milder form presenting with only myopathy. The majority of the late-onset patients benefit from riboflavin therapy[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The genetic background of late-onset MADD is heterogeneous and most of these patients carry variants in electron transfer flavoprotein dehydrogenase gene (\u003cem\u003eETFDH\u003c/em\u003e), while a minority have variants in the genes encoding the alpha (\u003cem\u003eETFA\u003c/em\u003e) and beta (\u003cem\u003eETFB\u003c/em\u003e) subunits of the electron transfer flavoprotein[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. It is reported that riboflavin transporter genes such as \u003cem\u003eSLC52A1\u003c/em\u003e, \u003cem\u003eSLC52A2\u003c/em\u003e and \u003cem\u003eSLC52A3\u003c/em\u003e [\u003cspan additionalcitationids=\"CR12 CR13 CR14\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], mitochondrial FAD transporter gene \u003cem\u003eSLC25A32\u003c/em\u003e [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] as well as flavin adenine dinucleotide synthase (FADS) gene genes \u003cem\u003eFLAD1\u003c/em\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] are related to riboflavin responsive MADD like phenotype[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecently, more cases with \u003cem\u003eFLAD1\u003c/em\u003e variants were reported as a cause of MADD, which presents heterogeneous response to riboflavin treatment depending on different genotype[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. We described here a patient with riboflavin responsive LSM and MADD resulting from \u003cem\u003eFLAD1\u003c/em\u003e gene variant (\u003cem\u003eFLAD1\u003c/em\u003e-RRMADD) and carried out studies on gene expression between \u003cem\u003eFLAD1-\u003c/em\u003eRRMADD and \u003cem\u003eETFDH-\u003c/em\u003eRRMADD to investigate why these two genes with different functions cause such similar phenotype. We reviewed literature and summarized 9 cases with riboflavin responsive \u003cem\u003eFLAD1\u003c/em\u003e gene variants, followed by comparing these 10 cases with 106 \u003cem\u003eETFDH-\u003c/em\u003eRRMADD cases in our neuromuscular center on clinical history, laboratory investigations and muscle pathology.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatient\u003c/h2\u003e \u003cp\u003eBetween 1995 and 2019 at Research Institute of Neuromuscular and Neurodegenerative Diseases, Qilu Hospital, Shandong University, a total of 106 patients with riboflavin responsive late-onset MADD were identified with \u003cem\u003eETFDH\u003c/em\u003e gene variants (\u003cem\u003eETFDH-\u003c/em\u003eRRMADD), only one case with riboflavin responsive late-onset MADD was proved with \u003cem\u003eFLAD1\u003c/em\u003e gene variants (\u003cem\u003eFLAD1\u003c/em\u003e-RRMADD)[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. This patient with \u003cem\u003eFLAD1\u003c/em\u003e gene variants has been followed-up regularly for over 7 years after he was diagnosed in 2015.\u003c/p\u003e \u003cp\u003eThis retrospective study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the medical Ethics Committee of Qilu Hospital (number: KYLL-202208-024). Informed consent for diagnostic muscle biopsy, research and publication was obtained from all individual participants included in the study (106 \u003cem\u003eETFDH-\u003c/em\u003eRRMADD and one \u003cem\u003eFLAD1\u003c/em\u003e-RRMADD).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eMuscle pathology\u003c/h2\u003e \u003cp\u003eOpen muscle biopsy in left biceps brachii was carried out under local anaesthesia. Muscle specimens were frozen in isopentane that was precooled in liquid nitrogen and stored at -80℃. For histological examination, serial frozen sections (8 mm) were stained with hematoxylin-eosin (H\u0026amp;E), Oil red O (ORO), succinate dehydrogenase (SDH), modified Gomori trichrome (mGT), nicotinamide adenine dinucleotide-tetrazolium reductase (NADH-TR), cytochrome c oxidase (COX) and periodic acid Schiff (PAS). For immunohistochemistry studies, serial frozen sections (5 mm) were incubated with dystrophin, dysferlin, α-sarcoglycan, β-sarcoglycan, γ-sarcoglycan, δ-sarcoglycan, and caveolin-3 antibodies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eTranscriptomics\u003c/h2\u003e \u003cp\u003eTotal amounts and integrity of RNA from muscle before riboflavin treatment were assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). Total RNA was used as input material for the RNA sample preparations. After the construction of the library, the library was initially quantified by Qubit2.0 Fluorometer, then diluted to 1.5ng/ul, and the insert size of the library is detected by Agilent 2100 bioanalyzer. After insert size meets the expectation, qRT-PCR is used to accurately quantify the effective concentration of the library to ensure the quality of the library. The different libraries are pooling according to the effective concentration and the target amount of data off the machine, then being sequenced by the Illumina NovaSeq 6000. The end reading of 150bp pairing is generated. The basic principle of sequencing is to synthesize and sequence at the same time (Sequencing by Synthesis). FeatureCounts (v1.5.0-p3) was used to count the reads numbers mapped to each gene. And then FPKM (Fragments Per Kilobase Million) of each gene was calculated based on the length of the gene and reads count mapped to this gene. Based on the gene expression level, the differentially expression genes (DEGs) between samples can be identified. With DEGs, we perform Gene Ontology (GO) classification, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway classification and functional enrichment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eGDF15 level detection in serum\u003c/h2\u003e \u003cp\u003eThe analysis of GDF15 level in serum was carried out according to the instructions of a human GDF15 ELISA Kit (ab155432, Abcam Plc, United Kingdom)\u003c/p\u003e \u003cp\u003e \u003cb\u003eLiterature review and comparation study\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe compared \u003cem\u003eFLAD1-\u003c/em\u003eRRMADD (our case together with cases review from literature) and \u003cem\u003eETFDH-\u003c/em\u003eRRMADD in our neuromuscular center (106 cases)[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStatistics\u003c/h2\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e1.Comparative study\u003c/h2\u003e \u003cp\u003eStatistical analysis was conducted with SPSS v.12.0 software package for Windows (SPSS Inc.), applying the paired Student's t test and two-sided Student's t test. The categorical variables were studied using Chi-square analysis. The numerical variables were studied using t-test. Two-sided p values were computed, and p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.Transcriptomics study\u003c/h2\u003e \u003cp\u003eFor DESeq2 with biological replicates: Differential expression analysis of two conditions/groups (two biological replicates per condition) was performed using the DESeq2 R package (1.20.0). DESeq2 provide statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The resulting P-values were adjusted using the Benjamini and Hochberg\u0026rsquo;s approach for controlling the false discovery rate. padj\u0026thinsp;\u0026lt;\u0026thinsp;=\u0026thinsp;0.05 and |log2(foldchange)|\u0026gt;=1 were set as the threshold for significantly differential expression.\u003c/p\u003e \u003cp\u003eFor edgeR without biological replicates: Prior to differential gene expression analysis, for each sequenced library, the read counts were adjusted by edgeR program package through one scaling normalized factor. Differential expression analysis of two conditions was performed using the edgeR R package (3.22.5). The P values were adjusted using the Benjamini \u0026amp; Hochberg method. padj\u0026thinsp;\u0026lt;\u0026thinsp;=\u0026thinsp;0.05 and |log2(foldchange)|\u0026gt;=1 were set as the threshold for significantly differential expression.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eClinical features and follow up\u003c/h2\u003e \u003cp\u003eThe patient was 56-year-old male. His developmental milestones were normal. During childhood he was able to run as fast as his peers. The limb strength was normal before he was 51-year-old, when he had progressive muscle weakness on lower extremities. His upper extremities were involved one month ago. When consulted, he complained with difficulty in standing up from squatting position and lifting arms over his shoulder, as well as tiredness after walking for 100 meters and muscle tenderness. He didn\u0026rsquo;t describe any difficulty with drinking, chewing or swallowing. There was a past history of cigarettes smoking. Alcohol and substance use was denied. There was no family history of muscle disease and the pedigree was shown in Fig.\u0026nbsp;1A. Neurologic examination revealed that he was awake, alert and interactive. Vision and speech normal. Pupils are equal and reactive. Extraocular movement intact. Facial strength normal. No weakness on eye or mouth closure. Tongue movements normal. Motor strength using the medical research council (MRC) scale: Neck flexion and extension 5/5. deltoids 3/5, biceps 4/5(left) and 4+/5(right), triceps 5-/5(left) and 5/5(right), wrist extension and flexion 5/5, finger extension and flexion 5/5, hip flexion 4/5, knee and ankle extension or dorsiflexion 5/5. Muscle tone normal. No muscle atrophy. Sensory examination intact to pinprick, vibration, and position. Reflexes 2\u0026thinsp;+\u0026thinsp;symmetrical. He had continuously elevated creatine kinase(CK) fluctuating between 383 to 928 U/L (38\u0026ndash;174 U/L). Needle EMG showed myogenic pattern on deltoids and quadriceps; Sensory and motor nerves were normal; F wave, H reflex and repeating nerve stimulation were normal; No myotonic discharge. The ultrasound of heart, liver, gallbladder, pancreas, spleen and kidney as well as electrocardiogram was normal. The second generation sequencing using blood sample was carried out for gene analysis, and it revealed compound heterozygous variants in \u003cem\u003eFLAD1\u003c/em\u003e (c.1588C\u0026thinsp;\u0026gt;\u0026thinsp;T p.R530C reported previously[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]; c.1589G\u0026thinsp;\u0026gt;\u0026thinsp;C p.R530P ; Fig.\u0026nbsp;1B). Although DNA sample from the patient\u0026rsquo;s parents who passed away at an advanced age was not available, sequence analysis on Bam picture proved that variants c.1588C\u0026thinsp;\u0026gt;\u0026thinsp;T and c.1589G\u0026thinsp;\u0026gt;\u0026thinsp;C were from two isolated homologous chromosomes(Fig.\u0026nbsp;1C). Western blot against FADS (\u003cem\u003eFLAD1\u003c/em\u003e gene) in muscle sample before riboflavin treatment was significantly reduced in this patient (Supplemental material 1).\u003c/p\u003e \u003cp\u003eHis muscle strength was normal after 2 months treatment with riboflavin 80mg/day. Then he was placed on a reduction dose of riboflavin 30 mg/day in the following 7 years. During follow-up, his CK level, needle EMG on deltoids, the ultrasound examination of heart and electrocardiogram were normal. The riboflavin level in serum was 10.8 ng/ml (2.3\u0026ndash;14.6 ng/ml) after treatment. Acylcarnitine profile from dried blood spots revealed moderate elevations of C4-, C5-, C6-, C8-, C10-, C10:1-, C12-, C14:1-, C14:2- and C18:2-acylcarnitines. Urine organic acid analysis showed mild elevation of ethylmalonic acid. Muscle MRI showed muscle atrophy and fatty degeneration on the posterior thigh group on T2WI-FSE (Fast spin echo) after 5 years of riboflavin treatment, moreover, the fat infiltration signal was suppressed on T2WI-FSE-FS (Fat suppression) (Fig.\u0026nbsp;1D).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eMuscle pathology\u003c/h2\u003e \u003cp\u003eLight-microscopic assessment of histochemical stains showed a marked variation in fiber size due to a large number of small atrophic fibers. Fibers with centrally placed nuclei accouted for about 20%. A few highly atrophic fibers formed nuclear clumps (Black arrow in Fig.\u0026nbsp;2A). There were a lot of fibers with small round vacules (Stars in Fig.\u0026nbsp;2A) which were proved as type I fiber in ATPase staining (Fig.\u0026nbsp;2G and 2H). There were a few necrotic and regenerating fibers and atypical ragged red fibers(RRFs, Stars in Fig.\u0026nbsp;2B), as well as numerous COX-negative fibers(Stars in Fig.\u0026nbsp;2C). The ORO stain showed that lipid droplets were prominently increased predominantly in type I fibers (Fig.\u0026nbsp;2D). Diffuse SDH enzyme deficiency was noticed compared to normal control(Fig.\u0026nbsp;2E and 2F, separately). There was no group atrophy and the atrophic fibers are mainly type I (Fig.\u0026nbsp;2G and 2H). The immunohistochemistry studies did not show any abnormality.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eTranscriptomic study\u003c/h2\u003e \u003cp\u003eTo elucidate the underlying mechanisms of RRMADD, the whole transcriptome sequencing together with integrated bioinformatics analysis were conducted in this patient with \u003cem\u003eFLAD1-\u003c/em\u003eRRMADD, three patients with \u003cem\u003eETFDH-\u003c/em\u003eRRMADD and three normal controls. Venn diagram was used to display expressed gene between three groups shown in Fig.\u0026nbsp;3A. Summary of DEGs was shown in Fig.\u0026nbsp;3B. The results showed a total of 1610 DEGs between \u003cem\u003eFLAD1\u003c/em\u003e-RRMADD and control group, among these 709 up-regulated and 901 down-regulated DEGs were identified, involving numerous signaling pathways (Supplemental material 2\u0026thinsp;\u0026minus;\u0026thinsp;1); for that between \u003cem\u003eETFDH\u003c/em\u003e-RRMADD and control group, a total of 1143 DEGs, 623 up-regulated and 520 down-regulated DEGs were identified (Supplemental material 2\u0026ndash;2); for \u003cem\u003eFLAD1\u003c/em\u003e-RRMADD and \u003cem\u003eETFDH\u003c/em\u003e-RRMADD group, a total of 1019 DEGs, 354 up-regulated and 665 down-regulated DEGs were identified (Supplemental material 2\u0026ndash;3).\u003c/p\u003e \u003cp\u003eVolcanic maps showed that the expression of \u003cem\u003eGDF\u003c/em\u003e15 mRNA was overwhelmingly significant up-regulated in both \u003cem\u003eFLAD1\u003c/em\u003e-RRMADD vs control and \u003cem\u003eETFDH\u003c/em\u003e-RRMADD vs control group (Fig.\u0026nbsp;3C and 3D). We next detected the GDF15 protein level in serum in \u003cem\u003eFLAD1\u003c/em\u003e-RRMADD after riboflavin treatment (N\u0026thinsp;=\u0026thinsp;1) and \u003cem\u003eETFDH\u003c/em\u003e-RRMADD (before and after riboflavin treatment, N\u0026thinsp;=\u0026thinsp;5). The GDF15 level in serum was an obvious increase in \u003cem\u003eETFDH\u003c/em\u003e-RRMADD group before and after riboflavin treatment comparing to normal controls (N\u0026thinsp;=\u0026thinsp;10, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;3E). For \u003cem\u003eFLAD1\u003c/em\u003e-RRMADD, the GDF15 level in the only patient after riboflavin treatment was significantly higher than the average of 10 normal controls (862.75 vs 342.68). The protein expression of GDF15 in muscle samples before riboflavin treatment was proved significantly increased in both \u003cem\u003eFLAD1\u003c/em\u003e-RRMADD and two \u003cem\u003eETFDH\u003c/em\u003e-RRMADD patients (Supplemental material 1).\u003c/p\u003e \u003cp\u003eVolcanic maps also showed that the expression of \u003cem\u003eTRIB3\u003c/em\u003e and \u003cem\u003eKLHDC7B\u003c/em\u003e mRNA was significant up-regulated in both \u003cem\u003eFLAD1\u003c/em\u003e-RRMADD vs control and \u003cem\u003eETFDH\u003c/em\u003e-RRMADD vs control group (Fig.\u0026nbsp;3C and 3D). Nevertheless, the protein study for those two genes failed to support the results that were found in mRNA study.\u003c/p\u003e \u003cp\u003eGO classification for the co-expressed genes of \u003cem\u003eFLAD1\u003c/em\u003e-RRMADD vs control and \u003cem\u003eETFDH\u003c/em\u003e-RRMADD vs control showed that the main differences were fatty acid metabolism process (Fig.\u0026nbsp;3F). KEGG pathway classification analysis for the co-expressed genes of \u003cem\u003eFLAD1\u003c/em\u003e-RRMADD vs control and \u003cem\u003eETFDH\u003c/em\u003e-RRMADD vs control showed that the main differences were biosynthesis of amino acids and fatty acid metabolism (Fig.\u0026nbsp;3G).\u003c/p\u003e \u003cp\u003e \u003cb\u003eComparison between\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eFLAD1\u003c/span\u003e\u003cb\u003e-RRMADD and\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eETFDH\u003c/span\u003e\u003cb\u003e-RRMADD\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe reviewed literature and listed a summary of 18 cases with MADD/GAII caused by \u003cem\u003eFLAD1\u003c/em\u003e variants including the present case (Supplemental material 3), in which 10 patients were proved with full or partial riboflavin responsiveness(Full riboflavin responsiveness: After riboflavin treatment, the symptoms including muscle weakness disappear and the patient returns to a normal life. Partial riboflavin responsiveness: After riboflavin treatment, the symptoms are partially improved, the patient' condition is stable but not normal) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan additionalcitationids=\"CR20 CR21 CR22\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The comparison between 10 \u003cem\u003eFLAD1\u003c/em\u003e-RRMADD (9 cases from literature and the present case) and 106 \u003cem\u003eETFDH\u003c/em\u003e-RRMADD (in our neuromuscular center) was shown in Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe comparison for clinical, biochemical and pathological characteristics between \u003cem\u003eFLAD1\u003c/em\u003e-RRMADD and \u003cem\u003eETFDH\u003c/em\u003e-RRMADD\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMADD/GAII patients\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eFLAD1\u003c/em\u003e(10 cases)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eETFDH\u003c/em\u003e(106cases)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ep value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSex(M:F)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5:5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e61:45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOnset age, mean (range)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14.73y(2m-51y)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29.2y(5y-73y)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSymmetric limb girdle weakness\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8/10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e106/106\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCraniofacial and neck muscle weakness\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7/10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e89/106\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.26\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVomiting\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4/10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23/106\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.19\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMyogenic pattern in EMG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4/5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28/53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.24\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCK(U/L), mean(range)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2044.2\u003c/p\u003e \u003cp\u003e(380\u0026ndash;6612)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1504.8\u003c/p\u003e \u003cp\u003e(102-11022)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMuscle pathology\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLipid storage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7/7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e106/106\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAtypical RRF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1/7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e84/106\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDecreased SDH activity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2/7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e69/106\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.52\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFaint COX staining\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4/7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0/106\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHomozygous mutation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7/10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8/106\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eCK, serum creatine kinase (normal value: 26\u0026ndash;178 mmol/l);\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe onset age of \u003cem\u003eFLAD1\u003c/em\u003e-RRMADD ranges from birth to 51 years, in which 6 out of 10 cases presented in infancy, in contrast, cases with \u003cem\u003eETFDH\u003c/em\u003e-RRMADD tend to be late-onset. On muscle pathology, 100% of \u003cem\u003eFLAD1\u003c/em\u003e-RRMADD or \u003cem\u003eETFDH\u003c/em\u003e-RRMADD were proved with lipid storage myopathy. The atypical RRFs were more frequent in \u003cem\u003eETFDH\u003c/em\u003e-RRMADD, while the fibers with faint COX staining were more common in \u003cem\u003eFLAD1\u003c/em\u003e-RRMADD (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, respectively). On muscle weakness symptom, accompanying symptoms such as vomiting, electromyography, serum CK level, and other muscle pathology features such as lipid storage or decreased SDH activity, it was similar between \u003cem\u003eFLAD1\u003c/em\u003e-RRMADD and \u003cem\u003eETFDH\u003c/em\u003e-RRMADD.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe described here a case with compound heterozygous \u003cem\u003eFLAD1\u003c/em\u003e-RRMADD (c.1588C\u0026thinsp;\u0026gt;\u0026thinsp;T p.R530C and c.1589G\u0026thinsp;\u0026gt;\u0026thinsp;C p.R530P in the FADS domain), and followed-up this patient for 7 years. The patient with \u003cem\u003eFLAD1\u003c/em\u003e variants was characterized by late-onset progressive proximal muscle weakness, lipid storge myopathy and dramatic good responsiveness to riboflavin treatment, which were extremely similar to \u003cem\u003eETFDH\u003c/em\u003e-RRMADD. After this patient was clinically improvement, it was proved that his plasma acylcarnitine was still abnormal featured by elevated short, medium and long chain acylcarnitine, as well as slightly increased ethylmalonic acid in urine organic acid. After riboflavin treatment for 5 years, although his muscle strength was normal, it was proved that this patient still had obvious muscle atrophy and fatty degeneration in posterior thigh group on muscle MRI, as reported previously in medial calf muscles[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo date, there were totally 17 MADD/GAII cases from 9 countries caused by \u003cem\u003eFLAD1\u003c/em\u003e variants according to the publications until April, 2022[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan additionalcitationids=\"CR20 CR21 CR22 CR23 CR24 CR25\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Among those cases, 9 patients were proved with full or partial riboflavin responsiveness. Then, in order to learn more information on \u003cem\u003eFLAD1\u003c/em\u003e-RRMADD, after comparing 10 \u003cem\u003eFLAD1\u003c/em\u003e-RRMADD (9 cases from literature and one case in our neuromuscular center) and 106 \u003cem\u003eETFDH\u003c/em\u003e-RRMADD (in our neuromuscular center), some new understanding on this disease was added. The \u003cem\u003eFLAD1\u003c/em\u003e-RRMADD could be infancy onset or late-onset. Except for lipid storage myopathy, the fibers with faint COX staining were more common in \u003cem\u003eFLAD1\u003c/em\u003e-RRMADD on muscle pathology. The proportion of consanguineous homozygotes was higher in \u003cem\u003eFLAD1\u003c/em\u003e-RRMADD. On the other clinical, biochemical or pathological features, it was similar between \u003cem\u003eFLAD1\u003c/em\u003e-RRMADD and \u003cem\u003eETFDH\u003c/em\u003e-RRMADD. It is hard to differentiate the two types of RRMADD being independent of genetic testing.\u003c/p\u003e \u003cp\u003eAmong 17 MADD/GAII cases caused by \u003cem\u003eFLAD1\u003c/em\u003e variants in publication, 9 cases were full or partially responsive to riboflavin treatment. Meanwhile riboflavin regimen was not administrated in 4 patients and was discontinued due to side effects in one patient, for this reason we are not sure whether those 5 patients could be cured by riboflavin treatment[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Only three patients with nonsense or frameshift variants (homozygous p.Arg249\u0026lowast;; p. Arg109Ala fs*3 and p.Ser167Pro fs*20; p.Glu266Val fs*3 and splice site variant) were reported as poor responsiveness to riboflavin treatment[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Whether to be responsive to riboflavin may be dependent on the genotype of \u003cem\u003eFLAD1\u003c/em\u003e variants. It is proved that the phenotype with frameshift variants seems more severe than that harboring single amino acid changes in the FADS domain[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], that may account for the different responsiveness to riboflavin in different patients. The fact that our case carried two heterozygous amino acid changes in FADS domain and was well responsive to single riboflavin treatment supported the theory above.\u003c/p\u003e \u003cp\u003eTranscriptomics study and GDF15 protein detection showed that the expression of \u003cem\u003eGDF15\u003c/em\u003e mRNA in muscle and protein in both serum and muscle was significant up-regulated in \u003cem\u003eFLAD1\u003c/em\u003e-RRMADD and \u003cem\u003eETFDH\u003c/em\u003e-RRMADD groups. GDF15 is a stress response cytokine that regulates energy metabolism. For now, we are unclear whether the increase of GDF15 is a cause or a result in RRMADD patients. The expression of GDF15 receptor, GFRAL/RET was not upregulated in this study suggesting that the increase of GDF15 might be a result of abnormal energy metabolism. On the other hand, it was reported that GDF15 could activate AMPK pathway in the absence of the GDF15 receptor GFRAL in skeletal muscle[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], and GDF15 is produced by skeletal muscle but targets adipose tissue to promote lipolysis[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], both of which may support that the increase of GDF15 is a cause of abnormal energy metabolism. Hence, future studies of the effects of GDF15 in lipid storage myopathy are highly needed. Gene enrichment analysis revealed that fatty acid metabolism was upregulated in both \u003cem\u003eFLAD1\u003c/em\u003e-RRMADD and \u003cem\u003eETFDH\u003c/em\u003e-RRMADD, which may result from negative feedback regulation involving in pathological process of lipid storage in skeletal muscle.\u003c/p\u003e \u003cp\u003eTranscriptomics study also showed that the expression of \u003cem\u003eTRIB3\u003c/em\u003e and \u003cem\u003eKLHDC7B\u003c/em\u003e mRNA was significant up-regulated in both \u003cem\u003eFLAD1\u003c/em\u003e-RRMADD and \u003cem\u003eETFDH\u003c/em\u003e-RRMADD except for \u003cem\u003eGDF15\u003c/em\u003e. We have done protein analysis and searched these genes function in NCBI database or Pubmed literature, also done analysis of protein protein interaction (PPI) network using STRING protein interaction database or Cytoscape software. After all these studies, only \u003cem\u003eGDF15\u003c/em\u003e is the most likely key gene that may involve in the pathogenesis of both \u003cem\u003eFLAD1\u003c/em\u003e-RRMADD and \u003cem\u003eETFDH\u003c/em\u003e-RRMADD. And an ongoing work was performing in our lab on \u003cem\u003eGDF15\u003c/em\u003e with a hybrid mice model of gdf15\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eetfdh\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e. We look forward to finding out something new.\u003c/p\u003e \u003cp\u003eOverall, we described a case with \u003cem\u003eFLAD1\u003c/em\u003e-RRMADD who has similar clinical, biochemical, and fatty acid metabolism changes to \u003cem\u003eETFDH\u003c/em\u003e-RRMADD, as well as the up-regulated expression of \u003cem\u003eGDF15\u003c/em\u003e gene in muscle and GDF15 protein in serum. A comparative study on \u003cem\u003eFLAD1-\u003c/em\u003eRRMADD and \u003cem\u003eETFDH-\u003c/em\u003eRRMADD revealed that COX deficiency fibers might be a pathological marker for differentiating \u003cem\u003eFLAD1\u003c/em\u003e-RRMADD from \u003cem\u003eETFDH\u003c/em\u003e-RRMADD.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors are grateful to the patient and his families for their participation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONFLICTOFINTEREST \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors report no conflict of interest related to this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY STATEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eETHICAL APPROVAL \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was approved by the Ethics Committee of Qilu Hospital, Shandong University (number: KYLL-202208-024).\u0026nbsp;Informed consent was obtained from all patients for being included in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING\u0026nbsp;\u003c/strong\u003eThis\u0026nbsp;work\u0026nbsp;was\u0026nbsp;supported\u0026nbsp;by\u0026nbsp;the\u0026nbsp;National\u0026nbsp;Natural\u0026nbsp;Science\u0026nbsp;Foundation\u0026nbsp;of\u0026nbsp;China\u0026nbsp;(No.82071412);\u0026nbsp;the Youth Program of National Natural Science Foundation of China (81701058，81901278); National Key Research and Development Program of China (2021YFC2700904); Qingdao Technology Program for Health and Welfare (20\u0026ndash;3-4\u0026ndash;42-nsh); the Taishan Scholars Program of Shandong Province.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eORCID ID\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBing Wen 0000-0001-8141-6662\u003c/p\u003e\n\u003cp\u003eTan Wang 0000-0002-1061-161X\u003c/p\u003e\n\u003cp\u003eChuanzhu Yan 0000-0002-2191-5184\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBruno C and Dimauro S Lipid storage myopathies. 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Patient with multiple acyl-CoA dehydrogenation deficiency disease and FLAD1 mutations benefits from riboflavin therapy. Neuromuscul Disord. 2017;27:581\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee YJ, Kim SY, Kim MJ, Kim AR, Lee JM, and Chae JH Infant with early onset bilateral facial and bulbar weakness: Successful treatment of riboflavin in multiple acyl-CoA dehydrogenase deficiency caused by biallelic nonsense FLAD1 variants. Neuromuscul Disord. 2021;31:1194\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVengalil S, Polavarapu K, Preethish-Kumar V, Nashi S, Arunachal G, Chawla T, et al. Mutation Spectrum of Primary Lipid Storage Myopathies. Ann Indian Acad Neurol. 2022;25:106\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMuru K, Reinson K, Kunnapas K, Lillevali H, Nochi Z, Mosegaard S, et al. FLAD1-associated multiple acyl-CoA dehydrogenase deficiency identified by newborn screening. Mol Genet Genomic Med. 2019;7:e915.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYamada K, Ito M, Kobayashi H, Hasegawa Y, Fukuda S, Yamaguchi S, et al. Flavin adenine dinucleotide synthase deficiency due to FLAD1 mutation presenting as multiple acyl-CoA dehydrogenation deficiency-like disease: A case report. Brain Dev. 2019;41:638\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGarcia-Villoria J, De Azua B, Tort F, Mosegaard S, Ugarteburu O, Texido L, et al. FLAD1, encoding FAD synthase, is mutated in a patient with myopathy, scoliosis and cataracts. Clin Genet. 2018;94:592\u0026ndash;3.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAguilar-Recarte D, Barroso E, Guma A, Pizarro-Delgado J, Pena L, Ruart M, et al. GDF15 mediates the metabolic effects of PPARbeta/delta by activating AMPK. Cell Rep. 2021;36:109501.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLaurens C, Parmar A, Murphy E, Carper D, Lair B, Maes P, et al. Growth and differentiation factor 15 is secreted by skeletal muscle during exercise and promotes lipolysis in humans. JCI Insight. 2020;5.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"lipid storage myopathy, riboflavin, multiple acyl-CoA dehydrogenation deficiency, FLAD1 gene, ETFDH gene, GDF15 gene","lastPublishedDoi":"10.21203/rs.3.rs-2314639/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2314639/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLipid storage myopathy (LSM) is a heterogeneous group of lipid metabolism disorders predominantly affecting skeletal muscle by triglyceride accumulation in muscle fibers. Riboflavin therapy has been shown to ameliorate symptoms in some LSM patients who are essentially concerned with multiple acyl-CoA dehydrogenation deficiency (MADD). It is proved that riboflavin responsive LSM caused by MADD is mainly due to \u003cem\u003eETFDH\u003c/em\u003e gene variant (\u003cem\u003eETFDH-\u003c/em\u003eRRMADD). We described here a case with riboflavin responsive LSM and MADD resulting from \u003cem\u003eFLAD1\u003c/em\u003e gene variants (c.1588C\u0026thinsp;\u0026gt;\u0026thinsp;T p.R530C and c.1589G\u0026thinsp;\u0026gt;\u0026thinsp;C p.R530P, \u003cem\u003eFLAD1-\u003c/em\u003eRRMADD). And we compared our patient together with 9 \u003cem\u003eFLAD1-\u003c/em\u003eRRMADD cases from literature to 106 \u003cem\u003eETFDH-\u003c/em\u003eRRMADD cases in our neuromuscular center on clinical history, laboratory investigations and pathological features. Furthermore, the transcriptomics study on \u003cem\u003eFLAD1-\u003c/em\u003eRRMADD and \u003cem\u003eETFDH-\u003c/em\u003eRRMADD were carried out. On muscle pathology, both \u003cem\u003eFLAD1\u003c/em\u003e-RRMADD and \u003cem\u003eETFDH\u003c/em\u003e-RRMADD were proved with lipid storage myopathy in which atypical ragged red fibers were more frequent in \u003cem\u003eETFDH\u003c/em\u003e-RRMADD, while fibers with faint COX staining were more common in \u003cem\u003eFLAD1\u003c/em\u003e-RRMADD. Molecular study revealed that the expression of \u003cem\u003eGDF15\u003c/em\u003e gene in muscle and GDF15 protein in both serum and muscle was significantly increased in \u003cem\u003eFLAD1-\u003c/em\u003eRRMADD and \u003cem\u003eETFDH\u003c/em\u003e-RRMADD groups. Our data revealed that \u003cem\u003eFLAD1-\u003c/em\u003eRRMADD (p.R530) has similar clinical, biochemical, and fatty acid metabolism changes to \u003cem\u003eETFDH\u003c/em\u003e-RRMADD except for muscle pathological features.\u003c/p\u003e","manuscriptTitle":"A Comparative study on riboflavin responsive multiple acyl-CoA dehydrogenation deficiency due to variants in FLAD1 and ETFDH gene","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-12-16 18:10:46","doi":"10.21203/rs.3.rs-2314639/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"047be275-f60f-4987-a65a-c0a54ff7fff5","owner":[],"postedDate":"December 16th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":17687672,"name":"Biological sciences/Genetics/Clinical genetics/Disease genetics"},{"id":17687673,"name":"Health sciences/Diseases/Neurological disorders/Neuromuscular disease"}],"tags":[],"updatedAt":"2023-03-07T13:20:58+00:00","versionOfRecord":[],"versionCreatedAt":"2022-12-16 18:10:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2314639","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2314639","identity":"rs-2314639","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}