Fatal multiple acyl-CoA dehydrogenase deficiency caused by ETFDH gene mutation: A case report

Abstract

BACKGROUND

Multiple acyl-CoA dehydrogenase deficiency (MADD) is a disease of rare autosomal recessive disorder. There are three types of MADD. Type I is a neonatal-onset form with congenital anomalies. Type II is a neonatal-onset form without congenital anomalies. Type III is considered to a milder form and usually responds to riboflavin. However, late-onset form could also be fatal and not responsive to treatments.

CASE SUMMARY

We report a severe case of a young man with onset type III MADD induced by drugs and strenuous exercise characterized by rhabdomyolysis and liver dysfunction. Urine analysis indicated 12 out of 70 kinds of organic acids like glutaric acid-2 were detected. Serum analysis in genetic metabolic diseases revealed 24 out of 43 tested items were abnormal, revealing the elevation of several acylcarnitines and the reduction of carnitine in the patient. By next generation sequencing technology for gene sequencing related to fatty acid oxidation and carnitine cycle defects, a rare ETFDH gene variant was identified: NM_004453:4:C.1448C>T(p.Pro483 Leu). The patient was diagnosed with late-onset GAII. He was not responsive to riboflavin and progressively worsened into multiple organ failure that finally led to death.

CONCLUSION

Type III MADD can also be fatal and not responsive to treatments.

Key Words: Electron transfer flavoprotein dehydrogenase mutation; Multiple acyl-CoA dehydrogenase deficiency; Multiple organ failure; Case report

Core Tip: Multiple acyl-CoA dehydrogenase deficiency (MADD) is a disease of rare autosomal recessive disorder of fatty acid, amino acid, and choline metabolism. Here, we report a severe case of a young man with onset type III MADD characterized by rhabdomyolysis and liver dysfunction. His urinary and serum analysis indicated organic acids, the elevation of several acylcarnitines and the reduction of carnitine. Eventually, we identified a rare compound heterozygous variant in the patient. Unfortunately, the patient was not responsive to riboflavin and his condition progressively worsened into multiple organ failure that finally led to death.

INTRODUCTION

Multiple acyl-CoA dehydrogenase deficiency (MADD), also called glutaric aciduria type II (GA II) (MIM:231680), is a rare autosomal recessive disorder. A defect in the alpha subunit of fatty acid mitochondrial electron transfer flavoprotein (ETFA) protein, beta subunit of fatty acid mitochondrial electron transfer flavoprotein (ETFB) protein or the electron transfer flavoprotein dehydrogenase (ETFDH) protein could lead to disorder of amino acid and choline metabolism. There are three types of MADD. Type I is a neonatal-onset form with congenital anomalies, Type II is a neonatal-onset form without congenital anomalies, and Type III is considered to a milder form and usually respond to riboflavin. Types I and II are usually considered to be more severe and sometimes fatal. The late-onset form is considered to be milder, and could be mostly responsive to treatments. Here, we report a late-onset MADD that is not responsive to riboflavin and is fatal.

CASE PRESENTATION

Chief complaints

Onset weakness for a month and vomiting for 5 h.

History of present illness

The patient complained of fatigue after strenuous exercise (tug of war) a month ago. He visited a doctor in a clinic and received a febuxostat tablet for hyperuricemia and bicyclol tablet for liver injury. He suffered, vomiting, shortness of breath, chest pain and slurred speech as the disease progressed.

History of past illness

The patient had a history of palpitation and liver dysfunction for 5 years.

Personal and family history

The patient denied infectious diseases, genetic diseases, history of surgical trauma and blood transfusion.

Physical examination

On admission, physical examination showed body temperature of 36.5 °C, pulse of 130 times per minute, respiration of 19 times per minute, and blood pressure [p(B)] of 16.226/10.374 kPa. The body check revealed jaundice, neck stiffness, flexor asthenia and weakness of four limbs.

Laboratory examinations

The patient’s blood glucose concentration was 2.7 mmol/L in the emergency room. Blood tests revealed an elevation of creatine kinase, alanine aminotransferase, aspartate aminotransferase on admission. His serum albumin was 37.2 U/L, serum creatinine was 80 μmol/L, and blood urea nitrogen was 6.4 mmol/L on admission, while his blood ammonia was 236 μmol/L, and ceruloplasmin was 13.1 mg/L. His urinalysis indicated occult blood in the urine. Blood clotting functions were abnormal. Blood cell analysis, index of infection, autoantibodies, tumor markers and thyroid function were normal on admission.

Imaging examinations

Computer tomography of the patient’s brain and abdomen indicted sallow cerebral sulci and severe fatty liver (Figure 1). Electrocardiograph indicated supraventricular tachycardia (SVT). The heart ultrasound and electromyography were normal.

Figure 1  Computer tomography indicted severe fatty liver and sallow cerebral sulci.

MULTIDISCIPLINARY EXPERT CONSULTATION

The patient was diagnosed with rhabdomyolysis, SVT, hepatic failure, acidosis, hypoglycemia and abnormal coagulation function on admission. A high dose of glucose was pumped in until his glucose level was stable. Timely fluid infusion, intravenous glutathione and glycyrrhzin were used for liver protection. Intravenous ornithine aspartate and rice vinegar clysis were applied for reducing blood ammonia level. However, the patient experienced very rapid deterioration in his clinical conditions following the onset of symptoms. His liver function and myocardial enzyme levels continuously increased. On the 9^th day after admission, he suffered from onset fatigue, mucocutaneous stained yellow (daily elevated serum bilirubin level over 17 mmol/L), repeated severe hypoglycemia, acidosis, hepatic encephalopathy, SVT, rhabdomyolysis, acute kidney injury, and respiratory failure (partial pressure of carbon dioxide of 10.241 kPa). He was then transferred to the intensive care unit.

His coagulation system, respiratory system, and circulatory system were collapsed, accompanied with renal and liver failure. Multiple organ failure could not be explained by the admitting diagnosis. Further, we discovered that his parents were in a consanguineous marriage. Combined with the clinical features of the patient, hereditary disease was considered. We then analyzed 70 types of organic acids in the patient’s urine, among which 12 organic acids were abnormally elevated. For example, glutaric acid -2 levels were 64 times higher than normal (Table 1). We then analyzed genetic metabolic diseases in amino acid and acylcarnitine spectrum in his blood. Twenty-four out of the total 43 tested items were abnormal, revealing the elevation of several acylcarnitines and the reduction of carnitine (Table 2), inferring MADD. Furthermore, we applied next generation sequencing technology for gene sequencing related to fatty acid oxidation and carnitine cycle defects in his whole blood. Single nucleotide variation and small fragment insertion deletion variation were detected, which indicated single nucleotide variation of ETFDH gene with chromosomal location: Chr4:159627503; mutation information: NM.004453.4:c.1448C>T(p.Pro483Leu); Homozygous, which indicated glutaric acidemia type II (MIM:231680).

Table 1 Organic acids tests in the urine.

Number	Organic acids (normal range)
1	Lactic acid -2 (0.0-13.0)	14.2↑
2	Pyruvate-OX-2 (0.0-30.0)	91.7↑
3	2-hydroxyisovaleric acid-2 (0.0-2.0)	7.8↑
4	2-ketoisovaleric acid-OX-2 (0.0-0.3)	1.0↑
5	2-ketoisohexanoic acid-OX-2 (0.0-0.8)	1.7↑
6	Glutaric acid -2 (0.0-8.0)	516.4↑
7	Isoamyl glycine-1 (0.0-1.5)	11.3↑
8	Acetylmalonic acid-2 (0.0-7.0)	17.6↑
9	Methylsuccinic acid-2 (0.0-4.0)	5.5↑
10	Methyl fumaric acid-2 (0.0-2.5)	3.0↑
11	Glutaconic acid-2 (0.0-0.0)	2.2↑
12	Adipic acid-2 (0.0-15.0)	165↑

12 of 70 organic acids were abnormal.

Table 2 Serum amino acid and acylcarnitine spectrum of genetic metabolic diseases.

Amino acid and acylcarnitine spectrum (normal range)		Amino acid and acylcarnitine spectrum
Palmitoyl carnitine (C16) (0.20-3.50)	4.49↑	C8/C3 (0.01-0.40)	1.20↑
Palmitoyl carnitine (C16:1) (0.02-0.30)	2.22↑	C10/C3 (0.01-0.50)	2.70↑
Palmolive dienyl carnitine (C16:2) (0.01-0.10)	0.31↑	C12/C3 (0.01-0.35)	3.58↑
Octadecyl carnitine (C18:1) (0.20-2.80)	3.80↑	C14/C3 (0.02-0.45)	9.25↑
C3/Mot (0.02-0.30)	0.01↓	C14:1/C8:1 (0.10-6.00)	12.31↑
C3DC/C4 (0.10-1.50)	0.07↓	C16/C2 (0.01-0.20)	0.82↑
C4/C2 (0.00-0.05)	0.08↑	C16/C3 (0.10-3.50)	22.07↑
C4/C3 (0.00-0.70)	2.15↑	C18/C3 (0.05-2.00)	5.44↑
C5/C2 (0.00-0.05)	0.12↑	C16 OH/C3 (0.00-0.15)	0.22↑
C5/C3 (0.00-0.50)	3.20↑	C18 OH/C3 (0.00-0.10)	0.17↑
C5DC/C3 (0.01-0.40)	0.65↑	(C1G+C18:1)/C2 (0.03-0.40)	1.52↑
C6/C3 (0.01-0.30)	0.72↑	C0/(C1G+C18) (6.50-90.00)	1.12↓

24 out of the total 43 tested items were abnormal, revealing the elevation of several acylcarnitines and the reduction of carnitine.

FINAL DIAGNOSIS

The patient was diagnosed with type III (late-onset form) MADD.

TREATMENT

Levocarnitine and riboflavin were intravenously injected at a dose of 60 mg/kg/d and 200 mg/d, respectively. Anti-infective therapy was applied for secondary infection. Unfortunately, the patient fell into a coma after the 9^th day of admission. He also suffered malignant arrhythmia with a heart rate over two hundred times per minute. Esmolol and metarbamine were not effective to his heart so synchronized electrical cardioversions were applied seven times on the 11^th, 12^th and 13^th day after his admission. The patients finally fell into a deep coma and depended on mechanical ventilation, continuous renal replacement treatment (CRRT) and artificial liver. Twenty-five days of ventilator support, eleven rounds of CRRT, two rounds of plasma exchange, three rounds of dual plasma molecular adsorption system and nine rounds of alveolar wash were applied within the twenty-five days of his coma. More than 20000 mL of plasma, 12 units of red blood cells, and some blood platelet and cryoprecipitation were used during the treatment. His liver function, kidney function and functions of other systems were improving, as demonstrated by clinical biochemistry (Figure 2).

Figure 2 Changes of clinical biochemistry after treatment. A: Coagulation system: levels of thromboplastin time (APTT), thrombin time (TT), prothrombin time (PT) and international normalized ratio (INR); B: Liver function: levels of alanine transaminase (ALT), aspartic transaminase (AST), direct bilirubin (DBIL), indirect bilirubin (IBIL); C: Kidney function and myolysis: levels of serum creatinine (Scr), creatine kinase (CK), lactate dehydrogenase (LDH-L), hydroxybutyrate dehydrogenase (HBDH); D: Infection indicators: levels of procalcitonin (PCT), high sensitive C-reactive protein (CRP), white blood cells (WBC), platelets (PLT).

OUTCOME AND FOLLOW-UP

The young man finally came to after 25 days of treatment, though he was still critically ill. We transferred the patient to Yunnan according to the demand of his parents. Unfortunately, we got the message that he finally passed away in Yunnan due to malignant arrhythmia.

DISCUSSION

MADD is also called glutaric aciduria type II (GA II) (MIM:231680). This is a rare autosomal recessive disorder caused by defects in the mitochondrial electron transport chain, which is the electron transport from flavin adenine dinucleotide-containing dehydrogenases to coenzyme Q10. Intra-mitochondrial acyl-CoA dehydrogenation causes MADD.

There are three forms of defects. Defects in the ETFA (OMIM 231680), ETFB protein (OMIM 130410) or ETFDH proteins (OMIM 231675) could lead to disorder of amino acid and choline metabolism. ETFA (15q23-q25) encodes the alpha subunit of ETF, ETFB (19q13.3-q13.4) encodes the beta subunit of ETF, and ETFDH (4q32-q35) encoding ETF-ubiquinone oxidoreductase (ETFQO).

Thus, there are three forms of MADD. Both type I and type II are neonatal-onset forms; type I is associated with congenital anomalies, while type II is not. Types I and II are usually considered to be more severe and sometimes fatal, presenting with hypoketotic hypoglycemia, metabolic acidosis, cardiomyopathy, and hepatomegaly. Type III is a late-onset form which is considered to be milder, characterized by proximal myopathy. Episodic vomiting, encephalopathy, liver and renal impairment, and rhabdomyolysis may occur under catabolic stress[1].

ETFDH variants are the most common cause of the late-onset form[2], which[3] is considered to be more variable. There could be recurrent hypoglycemia, metabolic acidosis, vomiting, and muscle weakness during stress[4]. These atypical symptoms usually make differential diagnosis difficult. Exercise intolerance could be an atypical symptom. In our case, exercise and drugs may have induced rhabdomyolysis and liver dysfunction. Research has also indicated that a prolonged exercise test could be of diagnostic importance[5]. Intolerance for prolonged exercise in patients with anamnestic signs would be an important diagnostic procedure that might replace the fasting. Medication could be another inducer. There have been reports that an anti-TB medication could increase MADD metabolic profiles[6]. Considered as a frequent missed diagnosis, late-onset of MADD should get more attention in the clinic when patients present with rhabdomyolysis with exercise or drug inducement. Our patient presented rhabdomyolysis, SVT, hepatic failure, acidosis, hypoglycemia and abnormal coagulation function that finally turned into multiple organ failure. It reminded us that late-onset MADD is also deadly; we should not only focus on prenatal diagnosis, but also pay attention to late-onset cases.

Urinary organic acid profiles could indicate MADD, which is important for the diagnosis of GAII. The test is characterized by elevated acids, including isovaleric acid, a-methylbutyrate, ethylmalonic acid, isobutyrate, glutaric acid, aliphatic dicarboxylic acids, etc[7,8]. Urine organic acid analysis could show these acids and their derivatives. During metabolic decompensation, lactate levels and creatine phosphokinase are typically raised. In our case, organic acids tests in the urine indicated 12 of 70 organic acids were abnormal, including lactic acid-2, pyruvate-OX-2, 2-hydroxyisovaleric acid-2, 2-ketoisovaleric acid-OX-2, etc. Among which, the patient’s glutaric acid-2 level was over 64 times higher than the upper limit of the reference level. Adipic acid-2 level was over 11 times higher than the upper limit of the reference level. Detection of urine organic acids are effective in MADD diagnosis that could highly suggest the disease.

Serum characterization of MADD includes acylcarnitine and organic acid profiling. The biochemical test would reveal higher levels of acylglycine conjugates and dicarboxylic acids. The elevated levels of C4-C18 acylcarnitines could be also detected in the blood. All these biochemical findings might normalize during the period of stability in MADD. Acylcarnitine profile could reveal elevations of short-chain, medium-chain and long-chain acylcarnitines[9]. In our case, blood amino acid and acylcarnitine spectrum of genetic metabolic diseases revealed that 24 out of the total 43 tested items were abnormal, including the elevation of several acylcarnitines and the reduction of carnitine.

Defects in the ETFDH protein[10] account for about 90% of MADD cases. ETFDH-c.250G>A is one of the most common mutations, representing a high allelic frequency of about 80% in southern China[11]. ETFDH-c.770A>G and ETFDH-c.1227A>C are more widespread mutations in mainland China[12]. Muscle biopsy could reveal acid and lipid accumulation in skeletal muscles, reducing the activity of mitochondrial respiratory chain complexes[13,14]. In our case, we applied next generation sequencing technology for gene sequencing related to fatty acid oxidation and carnitine cycle defects in the patient’s whole blood. Single nucleotide variation and small fragment insertion deletion variation were detected, which indicated single nucleotide variation of ETFDH gene with Chromosomal location: Chr4:159627503; mutation information: NM.004453.4:c.1448C>T(p.Pro483Leu). The mutation is very rare, and whether it is related to the severity of clinical symptoms could be further explored.

For treatments, type III MADD usually responds to riboflavin, with a reported response rate over 98%[1]. When riboflavin insufficient[15], MADD patients would undergo rapid degradation. Riboflavin supplementation is vital in the treatment of late-onset MADD. Research suggests that ETFDH mutations were responsible for all riboflavin-responsive GA-II patients[16]. More than 80% of patients treated with riboflavin showed a release of the disease. The treatment improved their cardiac and skeletal muscle functions. Patients receiving riboflavin treatment could also respond in urinary organic acids normalization and acylcarnitine species reductions[17]. However, there are controversies surrounding the treatment dose and course. No systematic evaluations have been reported yet. Unlike most late-onset GA-II patients, our patient did not respond to riboflavin treatment. Large amounts of riboflavin did not improve his condition, though he partially responded during the treatment. Whether the mutation was responsible for the poor outcome is worth further study.

In clinic, MADD should be suspected in cases with metabolic abnormalities. Patients with liver damage, kidney failure, hypoglycemia, hyperammonemia, hypoglycemia and rhabdomyolysis, etc would suggest GA-II. Exercise and medicine might be inducements. Early diagnosis depends on urine organic acid analysis, blood amino acid tests, serum acylcarnitine profile analysis, muscle biopsy, and genetic tests. Riboflavin supplementation is the first line medication, though it may be not effective.

CONCLUSION

In summary, our report describes the clinical, biochemical, and molecular findings of a 21-year-old male with ETFDH-related MADD. Late onset of MADD could be with definite inducement factors like drugs and severe exercise. It could also be fatal. c.1448C>T in ETFDH were found in this young man presented with multiple organ failure, which further expands the list of mutations found in MADD patients that might be riboflavin non-responsive MADD.

ACKNOWLEDGEMENTS

The authors are grateful to Dr. Xiao-Yong Zhang, Wan-Fang Tan and Fu-Ren Guan for entering the data.