Significant clinical heterogeneity with similar ETFDH genotype in three Chinese patients with late-onset multiple acyl-CoA dehydrogenase deficiency

Abstract

Late-onset multiple acyl-CoA dehydrogenase deficiency (MADD) with electron transfer flavoprotein dehydrogenase (ETFDH) gene mutations is the most common lipid storage myopathy (LSM) in China. Its clinical features vary widely and pose a challenge for diagnosis. We presented the significant clinical heterogeneity among three Chinese late-onset MADD patients with similar ETFDH genotype by collecting clinical information, muscle histology, and genetic analysis. Three novel compound heterozygous variants of ETFDH gene were identified: c.892C > T (p.Pro298Ser), c.453delA (p.Glu152ArgfsTer15), and c.449_453delTAACA (p.Leu150Ter). Moreover, all patients carried a hotspot mutation c.250G > A (p.Ala84Thr). Western blot analysis of the patients' muscular tissue showed a significantly reduced ETFDH expression, and normal electron transfer flavoprotein A (ETFA) and electron transfer flavoprotein B (ETFB) expression. Two patients with similar genotypes (c.453delA and c.449_453delTAACA) presented a significant clinical heterogeneity. Among them, one exhibited muscle weakness and exercise intolerance as initial and major symptoms, and the other showed episodic recurrent gastrointestinal symptoms before a serious muscle weakness appeared in later life. The novel variants in ETFDH and the corresponding clinical features enrich the variant spectrum of late-onset MADD and provide a new insight into the genotype-phenotype relationship. Late-onset MADD should be included in differential diagnosis for adult myopathy along with chronic digestive disease.

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Web End = Signicant clinical heterogeneity with similar ETFDH genotype in three Chinese patients with late-onset multiple acyl-CoA dehydrogenase deciency

Hong-xia Fu1 Xin-yi Liu1 Zhi-qiang Wang1,2 Ming Jin1 Dan-ni Wang1

Jun-jie He1 Min-ting Lin1 Ning Wang1,2

Received: 18 October 2015 / Accepted: 4 March 2016 / Published online: 21 March 2016 Springer-Verlag Italia 2016

Abstract Late-onset multiple acyl-CoA dehydrogenase deciency (MADD) with electron transfer avoprotein dehydrogenase (ETFDH) gene mutations is the most common lipid storage myopathy (LSM) in China. Its clinical features vary widely and pose a challenge for diagnosis. We presented the signicant clinical heterogeneity among three Chinese late-onset MADD patients with similar ETFDH genotype by collecting clinical information, muscle histology, and genetic analysis. Three novel compound heterozygous variants of ETFDH gene were identied: c.892C [ T (p.Pro298Ser), c.453delA(p.Glu152ArgfsTer15), and c.449_453delTAACA(p.Leu150Ter). Moreover, all patients carried a hotspot mutation c.250G [ A (p.Ala84Thr). Western blot analysis of the patients muscular tissue showed a signicantly reduced ETFDH expression, and normal electron transfer avoprotein A (ETFA) and electron transfer avoprotein B (ETFB) expression. Two patients with similar genotypes(c.453delA and c.449_453delTAACA) presented a significant clinical heterogeneity. Among them, one exhibited muscle weakness and exercise intolerance as initial and major symptoms, and the other showed episodic recurrent gastrointestinal symptoms before a serious muscle weakness appeared in later life. The novel variants in ETFDH and the corresponding clinical features enrich the variant

spectrum of late-onset MADD and provide a new insight into the genotype-phenotype relationship. Late-onset MADD should be included in differential diagnosis for adult myopathy along with chronic digestive disease.

Keywords Late-onset multiple acyl-CoA dehydrogenase deciency Lipid storage myopathy ETFDH Variants

Clinical heterogeneity

Introduction

Lipid storage myopathy (LSM) is due to inborn defects of fatty acid oxidation (FAO) and invariably characterized by accumulation of lipid droplets in myobers [1]. The biochemical deciencies and genetic causes of LSM include four disorders: multiple acyl-CoA dehydrogenase deciency (MADD); primary carnitine deciency (PCD); neutral lipid storage disease with ichthyosis (NLSDI); and neutral lipid storage disease with myopathy (NLSDM) [2].

MADD (OMIM 231680), also known as glutaric acid-uria type II (GA II), is an autosomal recessive disorder of fatty acid, amino acids, and choline metabolism. The phenotype of this disorder varies widely from a neonatal-onset form with/without congenital anomalies to a milder late-onset form, and is caused by mutation of electron transfer avoprotein A (ETFA), electron transfer avo-protein B (ETFB) or electron transfer avoprotein dehydrogenase (ETFDH) genes. Interestingly, most of patients with late-onset MADD are dramatically responsive to riboavin treatment and called riboavin-responsive MADD (RR-MADD), although its mechanism is still unknown. ETFDH deciency is the major causes of RRMADD [3]. To date, more than 80 missense ETFDH mutations have been reported and widely distributed in the

H. Fu and X. Liu contributed equally to this work.

& Ning Wang [email protected]

1 Department of Neurology and Institute of Neurology, First Afliated Hospital, Fujian Medical University, 20 Chazhong Road, Fuzhou 350005, China

2 Fujian Key Laboratory of Molecular Neurology, Fuzhou, China

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around the vessels without lipid accumulation in a local hospital. The Oil Red O (ORO) staining was not examined at that time. A working diagnosis of polymyositis was made and standard prednisolone (60 mg per day) therapy was commenced with transient and unsustained improvement. In view of signicant ongoing symptoms of myalgia and weakness, he was admitted to our hospital for re-examination and repeated muscle biopsy. His prenatal history and early development were normal. His life history showed the basis of poor physical tness, such as often catching a fever, vomiting and diarrhea after eating meat.Neurological examination revealed wadding gait, difcult in getting up from a sitting squatting position, positive Gowers sign, decreased tendon reexes, prominent neck and proximal limb weakness. The results of manual muscle testing (MMT) according to the Medical Research Council (MRC) for muscle strength were showed in Table 1.

Patient 2

A 15-year-old boy, the only child in his family, recognized proximal limbs weakness, fatigability, abdominal discomfort, and diarrhea with meat since he was about 5 years old.After that the symptoms had been relapsing and remitting about one to three times per year. And the symptoms were spontaneous recovery every time, but the mild exercise intolerance and weakness persisted and predominantly aggravated during infection and after intense activity. Half a year ago he relapsed with increasing myalgia and weakness. At admission, the patient revealed that his developmental milestones, past medical history of noted and dietary life were normal. Physical examination showed bilateral paraspinal atrophy, wadding gait, muscle weakness in proximal limbs, neck and trunk with decrease tender reexes (the MMT scale showed in Table 1). We did not observe winging scapula, muscle tenderness, pseudohypertrophy or hypotrophy except for percussive myotonia in the proximal and distal limbs.

Patient 3

A 68-year-old male was characterized by chronic or recurrent abdominal pains (RAP) or discomfort along with alter bowel function for over 10 years. When he was 60, he

Table 1 Manual muscle testing (MMT) scale of the three patients on examination

No. Neck Proximal upper limbs Proximal lower limbs Distal limbs

Flexion Extensor Deltoid Biceps Triceps Iliopsoas Gluteus medius Gluteus maximus

1 2? 3- 3? 4- 4? 3? 3? 3? 5

2 3 4 4 4? 5- 4- 4- 4? 5

3 3? 3? 4 5 5 3? 3 3 5

FAD, 4Fe4S, and ubiquinone (UQ) functional domains of the ETFDH protein. As previously reported, late-onset MADD due to ETFDH mutations is the most common form of LSM in China, but the variant spectrum and widespread hotspot mutations are distributed in different regions [4]. Three hotspot mutations were identied: c.250G [ A(p.Ala84Thr, rs121964954) in patients from southern China, and c.770A [ G (p.Try257Cys, rs780015493) andc.1227A [ C (p.Leu409Phe, CM095718) in patients from northern China [47].

Late-onset MADD shows clinical heterogeneity such as muscle weakness, acidosis, gastrointestinal symptoms, encephalopathy, hypoglycemia, and multisystem dysfunction. There is no clear genotype-phenotype relationship between patients with pure myopathy and metabolic crisis dominated. And the acylcarnitine prole and urinary organic acid analysis often show mild or atypical elevation, even decreased level [4, 8]. Thus, it posed a challenge to diagnosis. Here, we presented the signicant clinical heterogeneity in three late-onset MADD patients with similar genotype, including a special case of extremely late-onset MADD characterized by episodic recurrent gastrointestinal symptoms followed by progressive myopathy, in whom 3 novel compound heterozygous ETFDH variants were identied. These late-onset MADD-associated clinical features and novel variants enrich and provide a new insight into the genotype-phenotype relationship.

Materials and methods

Patients and clinical history

Patient 1

A 23-year-old male presented with 5-month of progressive muscle weakness with intermittent nauseating and vomiting in the morning. He suffered subacute premature fatigue, exercise intolerance, and weakness of shoulder, hip, neck, and masticatory muscles, general malaise and myalgia resembling polymyositis. He underwent a quadriceps muscle biopsy showing none specic myogentic change including a few inammatory cells inltrating

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received a rst diagnosis of irritable bowel syndrome (IBS) and was put on proton pump inhibitors (PPI) for a long time with poor clinical outcome. He kept on eld labor without any complaining of fatigability and muscle weakness during these years. At the age of 67, he experienced intermittent abdominal distension, nauseating and vomiting along with RAP and was transferred to gastroenterology department of a top hospital. Abdominal ultrasonography showed normal liver size, and grade I fatty change were presented. Electronic gastroscopy only revealed chronic gastritis without infection of Helicobacter pylori. Intestines mucous membranes showed no pathological change by colonoscopy examination. However, routine biochemical analysis showed remarked increases in muscle enzymes, serum myoglobin at 398.3 lg/L, without impaired renal function and myoglobinuria. The sustained hyperCKemia (about 2000 IU/L) lacked of rational explanation. The prediagnosis of intestine adhesion was made and indwelling gastric tube was performed to relieve abdominal dis-tension. After clinical symptomatology stabilized, he discharged home. Three months before the current admission, he felt gradually proximal limb and truncal muscle weakness, fatigue, myalgia, dysphagia, exertional and nocturnal dyspnea, and choking. This neuromyopathic symptomatology progressed rapidly associated with the IBS-like symptoms. At admission, he was wheelchair-bound and difcult to raise head due to severe muscle weakness and loss of appetite for a time. He had no other affected siblings, except one younger sister with similar IBS-like symptoms died of a head attack in her fties. Neurological examination revealed proximal muscle weakness, myalgia and dysarthria associated with obvious percussive myotonia (the MMT scale showed in Table 1).

The above patients came from the Han ethnic group in southern China and were not related to each other. Their parents were not consanguineous and had no myopathic symptom. Besides their clinical history, other clinical details including clinical features, MMT results, muscle enzymes, blood acylcarnitine and urine organic acids spectrum, electromyography (EMG), muscle magnetic resonance imaging (MRI), pulmonary function, electrocardiogram (EEG) and echocardiography were collected. After informed consent, peripheral blood and muscular tissue were obtained before riboavin treatment. Muscle tissue section staining including hematoxylin-eosin (HE), Modied Gomori Trichrome (MGT), NADH, PAS, ATP, and ORO were performed in our hospital. After diagnosed as late-onset MADD by muscle biopsy and gene sequencing, all the patients were treated with riboavin (6090 mg/day) and coenzyme Q10 (60 mg/day), and kept follow-up for more than half a year. This study was approved by the local ethical committee.

PCR and Sanger sequencing

DNA was isolated from peripheral blood samples (Qia-gene, Hilden, Germany). The coding exons and intron exon boundaries of ETFA, ETFB and ETFDH were amplied by polymerase chain reaction (PCR). The products were puried and sequenced using an ABI 3730XL Automated DNA Sequencer (PE Applied Biosystems, Foster City, CA, USA). The primers and amplifying conditions were based on a previously reported literature [6].

Bioinformatic analysis

The protein function prediction of novel variants were performed by SIFT (Sorting Intolerant From Tolerant) (http://sift.jcvi.org/www/SIFT_enst_submit.html

Web End =http://sift.jcvi.org/www/SIFT_enst_submit.html ), and Mutation Taster (http://www.mutationtaster.org/

Web End =http://www.mutationtaster.org/ ).

Western blot analysis of muscular ETFDH protein

The muscular tissues were homogenized and dissolved in 1 9 cell lysis buffer (Cell Signaling Technology; USA). After quantied (Bio-Rad Protein Assay Kit, USA), 200 lg muscular protein of each patients were separated by electrophoresis using 10 % sodium dodecyl sulfatepolyacrylamide gels. Nitrocellulose membranes (Whatman; UK) were probed with ETFDH antibody (ProteinTech Group, USA) and visualized with a rabbit IgG antibody (ZSGBBIO, Beijing, China) by ECL Western blotting detection system (Beyotime, Jiangsu, China). GAPDH (Beyotime, Jiangsu, China) was selected as internal control. The average grayscale of the bands was analyzed by Quantity One 4.6.2 software (Bio-Rad Laboratories, Sundbyberg, Sweden).

Results

Clinical information

All the three patients showed exercise intolerance, symmetric proximal muscle weakness, and difculty in lifting head as major clinical manifestation. Gastrointestinal symptoms such as vomiting and diarrhea also appeared. Amyotrophy, mastication decits, myalgia, and dysphagia appeared on part of the patients. Muscle enzymes including CK, LDH, and AST were higher than upper limit of normal (upper limit of normal: CK 140 U/L, LDH 245 U/L, and AST 40 U/L). Among the three patients, EMG showed myopathic changes, but normal nerve conduction and response to repetitive stimuli. Fatty liver was detected in patient 1 and patient 3. All the patients were performed muscle MRI of bilateral leg, and only the patient 3 showed

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diffuse atrophy with fat inltration in the inside muscle groups of bilateral hamstrings and gluteus maximus (Fig. 1). Lung function tests, compared to predicted values, was slightly reduced (FVC 76.1 % and FEV1 84.5 %) in patient 3, and was normal in other patients. EEG and echocardiography showed no cardiomyopathy. HE staining of the muscle tissue sections showed vacuoles distributing in the cytoplasm of myobers. ORO staining displayed the lipid droplets deposition in the myobers as our reported before [6]. The clinical features were summarized in Table 2. The spectrums of blood acylcarnitine and urine organic acids before treatment were showed in Table 3.

After 1 month riboavin treatment, all the patients showed a dramatic improvement in clinical symptom. At a 6-month follow-up, they were found to be totally symptom-free. The muscle strength and enzymes recovered close to normal levels.

Variants

Three novel mutations of c.892C [ T (p.Pro298Ser),c.453delA (p.Glu152ArgfsTer15), and c.449_453delTAACA

1M23??-???-480102474-Myogenicc.250G[A,c.892C[T

2M15????---29231739?Myogenicc.250G[A,c.453delA

3M68?????-?902436156?Myogenicc.250G[A,c.449-

453delTAACA

EMGGenotype

Fatty

liver

AST

(U/L)

ClinicalmanifestationExamination

Muscle

LDH

(U/L)

(U/L)

MyalgiaDysphagiaCK

Mastication

decits

AmyotrophyVomiting/

diarrhea

Upperlimitofnormal:CK140U/L,LDH245U/L,andAST40U/L

CKcreatininekinase,LDHlactatedehydrogenase,ASTaspartateaminotransferase,EMGelectromyography,Mmale,?positive,-negative

Exercise

intoleranee

weakness

Fig. 1 T1-weighted images (a, c, e) and fat suppression (T2-weighted short tau inversion recovery, T2-STIR) images (b, d, f) showed diffuse atrophy with fat inltration in bilateral hamstrings and gluteus maximus from patient 3 on coronal scanning (a, b), horizontal scanning of pelvic (c, d) and thigh (e, f) level

Table2Clinicalfeaturesofthethreepatientswithlate-onsetMADD

No.SexOnsetage

(years)

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Table 3 Blood acylcarnitine and urine organic acids spectrum of the three patients

No. Blood acylcarnitine spectrum Urine organic acids spectrum

1 C6, C8, C8DC, C10, C12, C12DC, C14, C14:1, C16:1, C16:2, C18, C18:2: Ethylmalonic acid

2 C4, C6, C5DC, C8, C10, C10:1, C12, C12:1, C12DC, C14, C14:1, C14:2, C16:1, C16:2, C18, C18:2: Normal

3 C12DC, C14, C14:1, C14DC, C16, C16:1, C18, C18:1:; C0, C2, C3; 4-Hydroxyphenyl-lactic acid

: The value of these data were above normal level

; The value of these data were below normal level

Fig. 2 Three novel variants located in ETFDH gene. The three novel variants c.892C [ T(p.Pro298Ser),c.449_453delTAACA(p.Leu150Ter), and c.453delA(p.Glu152ArgfsTer15) were conrmed by Sanger sequencing. The upper panel of each chromatogram described the wild-type sequence, and the lower panels presented the heterozygous varied sequence

(p.Leu150Ter) in ETFDH gene were identied (Fig. 2). These variants were not detected in 200 healthy controls from Han ethnic population in southern China after informed consent. The genotypes of the three patients were all compound heterozygous, and carried a hotspot variant c.250G [ A(p.Ala84Thr). Through bioinformatic analysis, the novel variants were precisely pathogenic. The SIFT, and Mutation Taster predicted that the three variants were possible disease causing. The three novel variants were not found in Exome Aggregation Consortium (ExAC). No variant was detected in ETFA or ETFB gene.

ETFDH protein expression in muscle

The c.892C [ T located on the UQ functional domain, and the two deletion variants located on the FAD functional domain of ETFDH [9]. Western blot analysis of muscular tissue of the three patients with late-onset MADD was performed. A control patient without hereditary metabolic disease and a late-onset MADD patient with c.250G [ A(p.Ala84Thr) homozygous variant were chose as negative control (C) and positive control (P0), respectively. A signicant reducing expression of ETFDH was detected among the four late-onset MADD patients, while that of negative control showed an afuent ETFDH protein

expression. There is no visible difference on the expression of ETFA and ETFB among the patients and control (Fig. 3).

Discussion

We report three novel mutations of ETFDH gene in southern Chinese patients with late-onset MADD, who presented clinical heterogeneity out of limb weakness and exercise intolerance. Among them, patient 2 and patient 3 shared similar genotypes (c.453delA and c.449_453del-TAACA) but showed a signicant clinical heterogeneity. The patient 2 exhibited muscle weakness and exercise intolerance as the major and initial symptom without signicant fat inltration by muscle MRI. While, patient 3 showed serious gastrointestinal symptoms before muscle weakness appeared, and muscle MRI showed fat inltration atrophy in bilateral hamstrings and gluteus maximus. The different symptoms and onset age among them who carried similar gene variants suggest the signicant clinical heterogeneity of late-onset MADD. The clinical features of our patients were quite different with that reported in Denmark [10], America [11], Japan [12], and other ethnicities [3]. In these populations, patients represented not

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Fig. 3 Western blot analysis of muscular ETFDH, ETFA, ETFB protein. Western blot analysis from patients with novel compound heterozygous variants (P1, P2, P3) and positive control with homozygous c.250G [ A (p.Ala84Thr) mutation of ETFDH (P0)

presented a signicantly reduced band at 200 lg level compared to negative control (C). C negative controls; P patient

only muscle weakness but also hypoglycemia, ammoniemia, hypotonia, encephalopathy, cardiomyopathy, and periodically metabolic acidosis, and the metabolic crisis was much more prominent than myopathy. Interestingly, the patients with serious metabolic symptoms showed a wide ETFDH variant spectrum without the hotspot variant. It is possible that a potential genotype-phenotype relationship exists between the metabolic dominant patients and patients with pure myopathy.

Even though clinical heterogeneity widely exists among late-onset MADD patients, some common physical signs could be found. First, the quadriceps femoris muscle strength is normal. It was not only observed on the three patients but also existed among other late-onset MADD patients [13]. Patient 1 once carried quadriceps femoris muscle biopsy, and the pathological result showed none specic myogenic change, which suggests that the quadriceps femoris is mildly involved. When the pathological result of quadriceps femoris showed no lipid droplet deposition, doctors need to make qualitative diagnosis according to clinical feature and gene analysis. Second, lung function tests of the patients were almost normal, which suggested the respiratory muscles were less affected. That is different with glycogen storage disease, which is easily involving breathing. Third, the obvious percussive myotonia was observed among the patients, but EMG did not show myotonic potentials. That is different with myotonic dystrophy and may be due to their different

mechanism. The symptoms of muscle weakness, exercise intolerance, amyotrophy, mastication decits, and myalgia are common in Chinese late-onset patients. The symptom of myopathy is necessary to differentiate from polymyositis, glycogen storage disease, mitochondrial disease, and progressive muscular dystrophy [13]. Meanwhile, in a few individual cases, just as the patient 3, the rst and dominant symptoms were serious gastrointestinal symptoms. It is easily misdiagnosed as digestive diseases. Some patients with high muscle enzymes and fatty liver were easily misdiagnosed as hepatopathy. These might lead to the delay of early diagnosis of late-onset MADD and riboavin treatment. So to late-onset MADD patients, we need to fully consider their overall clinical manifestations, including uctuating symptoms of the muscle, digestive tract, liver or other tissues.

We discovered 3 novel variants of ETFDH gene located in the FAD binding and UQ functional domain, and bioinformatics analysis showed that these novel variants were all pathogenic. The deletion variants might cause frame shift in ETFDH gene, lead to premature translation termination and produce truncated ETFDH protein. Our semi-quantitative analysis of western blot indicated a lower expression of ETFDH protein than negative control (Fig. 3). This result is consistent with the patients carryingc.250G [ A (p.Ala84Thr) homozygote mutation as our previous study [6], and the similar decrease expression of

ETFDH mutants has been conrmed by other groups [12, 14]. Furthermore, genotype-phenotype relationship suggested that the specic milder ETFDH variants of late-onset MADD might be responsible for the mild phenotype if there are different variants in the two alleles [3]. Compatible with it, each of our patients had one mild ETFDH mutation c.250G [ A (p.Ala84Thr), which likely accounts for their mild phenotype and respond to riboavin.

Although the pathogenic mechanism of MADD is unclear, instability of the ETFDH mutants due to misfolding has been considered as one of the major molecular causes. Riboavin works as a cofactor of ETFDH, which improves the conformation stability and reduces the degradation of ETFDH mutants [3, 9]. But the molecular mechanism of riboavin treating on late-onset MADD remains unknown and additional studies are necessary.

As we known, most of patients with late-onset MADD have a dramatic response to riboavin supplement (100400 mg/day) [15]. Up to now, some literatures reported that patients showed a mild recovery after glucocorticoids treatment [4, 16]. Here, patient 1 was prediagnosed as polymyositis and treated by glucocorticoids for more than 1 month. But the symptoms did not have a signicant improvement. Whether the treatment of glucocorticoids is working on late-onset MADD still needs quantitative analysis. On the other hand, the results with

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combination treatment by carnitine and coenzyme Q10 are controversial. Our patients had a good recovery after treatment with riboavin only combined with coenzyme Q10.

In conclusion, we identied three novel compound heterozygous mutations of ETFDH gene in patients with late-onset MADD, and discussed the signicant clinical heterogeneity among patients with similar genotype. Late-onset MADD should be included in the differential diagnosis for adult myopathy and chronic digestive disease. The novel mutations and their corresponding clinical features enrich the variant spectrum of late-onset MADD and provide a new insight into the genotype-phenotype relationship.

Acknowledgments We thank the patients and their family members for their cooperation. This work was supported by Grants from the National Natural Science Foundation of China (81271254, Beijing), National Key Clinical Specialty Discipline Construction Program, and Fujian Key Clinical Specialty Discipline Construction Program.

Compliance with ethical standards

Funding This study was funded by Grants from the National Natural Science Foundation of China (Grant Number: 81271254), National Key Clinical Specialty Discipline Construction Program, and Fujian Key Clinical Specialty Discipline Construction Program.

Conict of interest The authors declare that they have no conict of interest.

References

1. Burr ML, Roos JC, Ostr AJ (2008) Metabolic myopathies: a guide and update for clinicians. Curr Opin Rheumatol 20:639647

2. Ohkuma A, Noguchi S, Sugie H, Malicdan MC, Fukuda T, Shimazu K, Lpez LC, Hirano M, Hayashi YK, Nonaka I, Nishino I (2009) Clinical and genetic analysis of lipid storage myopathies. Muscle Nerve 39:333342

3. Olsen RK, Olpin SE, Andresen BS, Miedzybrodzka ZH, Pour-farzam M, Merinero B, Frerman FE, Beresford MW, Dean JC, Cornelius N, Andersen O, Oldfors A, Holme E, Gregersen N, Turnbull DM, Morris AA (2007) ETFDH mutations as a major cause of riboavin-responsive multiple acyl-CoA dehydrogenation deciency. Brain 130:20452054

4. Xi J, Wen B, Lin J, Zhu W, Luo S, Zhao C, Li D, Lin P, Lu J, Yan C (2014) Clinical features and ETFDH mutation spectrum in a

cohort of 90 Chinese patients with late-onset multiple acyl-CoA

dehydrogenase deciency. J Inherit Metab Dis 37:3994045. Wen B, Dai T, Li W, Zhao Y, Liu S, Zhang C, Li H, Wu J, Li D, Yan C (2010) Riboavin responsive lipid storage myopathy caused by ETFDH gene mutations. J Neurol Neurosurg Psychiatry 81:231236

6. Wang ZQ, Chen XJ, Murong SX, Wang N, Wu ZY (2011) Molecular analysis of 51 unrelated pedigrees with late-onset multiple acyl-CoA dehydrogenation deciency (MADD) in southern China conrmed the most common ETFDH mutation and high carrier frequency of c.250G [ A. J Mol Med 89:569576

7. Lan MY, Fu MH, Liu YF, Huang CC, Chang YY, Liu JS, Peng CH, Chen SS (2010) High frequency of ETFDH c.250G [ A mutation in Taiwanese patients with late-onset lipid storage myopathy. Clin Genet 78:565569

8. Wen B, Li D, Li W, Zhao Y, Yan C (2015) Multiple acyl-CoA dehydrogenation deciency as decreased acyl-carnitine prole in serum. Neurol Sci 36:853859

9. Zhang Jian, Frerman Frank E, Kim Jung-Ja P (2006) Structure of electron transfer avoprotein-ubiquinone oxidoreductase and electron transfer to the mitochondrial ubiquinone pool. Proc Natl Acad Sci USA 103:1621216217

10. Olsen RK, Pourfarzam M, Morris AA, Dias RC, Knudsen I, Andresen BS, Gregersen N, Olpin SE (2004) Lipid-storage myopathy and respiratory insufciency due to ETFQO mutations in a patient with late-onset multiple acyl-CoA dehydrogenation deciency. J Inherit Metab Dis 27:671678

11. Angle B, Burton BK (2008) Risk of sudden death and acute life-threatening events in patients with glutaric acidemia type II. Mol Genet Metab 93:3639

12. Yotsumoto Y, Hasegawa Y, Fukuda S, Kobayashi H, Endo M, Fukao T, Yamaguchi S (2008) Clinical and molecular investigations of Japanese cases of glutaric acidemia type 2. Mol Genet Metab 94:6167

13. Beresford MW, Pourfarzam M, Turnbull DM, Davidson JE (2006) So doctor, what exactly is wrong with my muscles? Glutaric aciduria type II presenting in a teenager. Neuromuscul Disord 16:269273

14. Olsen RK, Andresen BS, Christensen E, Bross P, Skovby F, Gregersen N (2003) Clear relationship between ETF/ETFDH genotype and phenotype in patients with multiple acyl-CoA dehydrogenation deciency. Hum Mutat 22:1223

15. Cornelius N, Frerman FE, Corydon TJ, Palmfeldt J, Bross P, Gregersen N, Olsen RK (2012) Molecular mechanisms of riboavin responsiveness in patients with ETF-QO variations and multiple acyl-CoA dehydrogenation deciency. Hum Mol Genet 21:34353448

16. Zhu M, Zhu X, Qi X, Weijiang D, Yu Y, Wan H, Hong D (2014) Riboavin-responsive multiple Acyl-CoA dehydrogenation deciency in 13 cases, and a literature review in mainland Chinese patients. J Hum Genet 59:256261

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