Hepatic Presentation of Late-Onset Multiple Acyl-CoA Dehydrogenase Deficiency (MADD): Case Report and Systematic Review

Diagnosis of pediatric steatohepatitis is a challenging issue due to a vast number of established and novel causes. Here, we report a child with Multiple Acyl-CoA Dehydrogenase Deficiency (MADD) presenting with an underrated muscle weakness, exercise intolerance and an atypically severe steatotic liver involvement. A systematic literature review of liver involvement in MADD was performed as well. Our patient is a 11-year-old otherwise healthy, non-obese, male child admitted for some weakness/asthenia, vomiting and recurrent severe hypertransaminasemia (aspartate and alanine aminotransferases up to ×20 times upper limit of normal). Hepatic ultrasound showed a bright liver. MRI detected mild lipid storage of thighs muscles. A liver biopsy showed a micro-macrovacuolar steatohepatitis with minimal fibrosis. Main causes of hypertransaminasemia were ruled out. Serum aminoacids (increased proline), acylcarnitines (increased C4-C18) and a large excretion of urinary glutaric acid, ethylmalonic, butyric, isobutyric, 2-methyl-butyric and isovaleric acids suggested a diagnosis of MADD. Serum acylcarnitines and urinary organic acids fluctuated overtime paralleling serum transaminases during periods of illness/catabolic stress, confirming their recurrent nature. Genetic testing confirmed the diagnosis [homozygous c.1658A > G (p.Tyr553Cys) in exon 12 of the ETFDH gene]. Lipid-restricted diet and riboflavin treatment rapidly ameliorated symptoms, hepatic ultrasonography/enzymes, and metabolic profiles. Literature review (37 retrieved eligible studies, 283 patients) showed that liver is an extramuscular organ rarely involved in late-onset MADD (70 patients), and that amongst 45 patients who had fatty liver only nine had severe presentation. Conclusion: MADD is a disorder with a clinically heterogeneous phenotype. Our study suggests that MADD warrants consideration in the work-up of obesity-unrelated severe steatohepatitis.


INTRODUCTION
Multiple acyl-CoA dehydrogenase deficiency (MADD, MIM #231680), also known as Glutaric aciduria type II, is a rare autosomal recessive inherited disorder of fatty acid, amino acid, and choline metabolism. It is caused by deficiency of either an electron-transfer flavoprotein (ETF, encoded by ETFA and ETFB genes) or an electrontransfer flavoprotein dehydrogenase (ETFDH, encoded by ETFDH gene). The metabolic defects result in impaired adenosine triphosphate (ATP) biosynthesis, excessive lipid accumulation in different organs and insufficient gluconeogenesis (1).
The genetic heterogeneity correlates with different clinical phenotypes that can be divided into three types: (1) neonatal onset with congenital anomalies (MADD type I): the symptoms appear during the first 24 h of life and patients usually die within the first week of life; (2) neonatal onset without anomalies (MADD type II), with symptoms arising within the first 24-48 h of life and the death often occurs within the first weeks of life; (3) mild and/or late onset (MADD type III) with course and age at presentation extremely variable; in adolescents and adults muscular or cardiac symptoms are usually first features suggestive for MADD (2). Extramuscular symptoms such as fatty liver and recurrent vomiting have been reported more rarely (3). Patients with late-onset MADD carry at least one missense variation with minor amounts of residual ETF/ETFDH activity. This seems to be sufficient to prevent embryonic development of congenital anomalies. Instead, homozygosity for null mutations is usually associated with MADD type I (1).
Diagnosis is based on both the urinary organic acids profile and the blood acylcarnitine pattern. Urine presents elevations of glutaric, lactic, ethylmalonic, adipic, suberic, sebacic, butyric, and isovaleric acids, with elevation of 2-hydroxyglutaric acid being considered pathognomonic. The plasma acylcarnitine profile demonstrates generalized elevations in most of short, medium, and long chain acylcarnitines such as C4, C5, C5-DC, C6, C8, C10, C12, C14:1, C16, and C18:1. Biochemical diagnosis of patients with type III MADD is often very challenging, because elevation of urine organic acids may be incomplete, subtle, intermittent, or elevated only during an acute metabolic crisis (4).
Here, we report a 12-year-old male child presenting some muscle weakness and exercise intolerance along with an unusual extremely severe recurrent liver involvement, which has hitherto been rarely reported in type III MADD. A systematic literature review of liver involvement in MADD has been performed as well.

CASE REPORT
The proband was a 12-year-old otherwise healthy child admitted for some weakness/asthenia along with episodes of vomiting, and recently discovered severe hypertransaminasemia [alanine aminotransferase (ALT) and aspartate aminotransferase (AST) × 20 times upper limit of normal (uln)]. He was born at term after a normally conducted pregnancy. Previous regular checkup of laboratory tests including transaminases were normal at age 1, 7, and 8 years old. His early neurodevelopment was reported as normal with walking at 1 year of age and talking in phrases at 18 months of age.
In June 2019, he was admitted to our ward for a new episode of vomiting, asthenia, and hypertransaminasemia and neutropenia. His physical examination included a weight of 37 kg (50-75th percentile), height of 146.8 cm (50-75th percentile) and body mass index of 17.2 (50th percentile). There was no facial dysmorphism or significant skin finding. Muscle mass was normal with normal strength and tone. No tremor, ataxia, or abnormal movements were present. At entry laboratory data confirmed hypertransaminasemia (ALT × 30 uln; AST × 20 uln; CK × 10 uln; LDH × 4 uln). Hepatic ultrasound showed a severe bright liver. Cardiac sonography was normal. Due to a persistent elevation of serum enzymes, a liver biopsy was performed and it showed a massive micro-macrovacuolar steatohepatitis picture with minimal fibrosis without signs of inflammation (Figures 1A,B).
During the hospitalization, main viral [Hepatitis A, B, and C and minor hepatotropic viruses serology (e.g., Epstein Barr Virus and Cytomegalovirus)], autoimmune [anti -nuclear, smooth muscle, liver and kidney microsomes, liver cytosol, endomysium, tissue transglutaminase antibodies], toxic and common metabolic (Wilson disease, hereditary fructose intolerance) causes of hypertransaminasemia were ruled out by appropriate tests and anamnesis. Blood glucose, gases, lactate, ammonia were repeatedly found normal. Asthenia was evaluated by 6 min walking test which resulted positive showing fatigue after only 1.49 min (with oxygen saturation 78%, heart frequency 180 beats per minute, meters traveled 160). Magnetic Resonance Imaging [1.5 Tesla Philips MR equipment, Turbo Spin Echo Fat Suppressed T2w technique (TR 7014 ms, TE 100 ms, FA 90 • ), using 5 mm thick slices and 2 excitations] was indicative of lipid storage of thighs muscles (mild, Figure 1C), and confirmed liver steatosis (severe) as well. At hospital discharge after 11 days CK was normal (112 U/L), AST × 1.2 uln; ALT × 4 uln; LDH × 2 uln.
The determination of urinary organic acids and the search for serum amino acids and acyl carnitines were requested. Serum aminoacids showed increased proline; most of serum acylcarnites were increased (C4-C18) (Supplementary Table 1). Urinary mass spectrometric analysis showed a large excretion of glutaric acid, ethylmalonic, butyric, isobutyric, 2-methylbutyric, and isovaleric acids suggesting a possible diagnosis of MADD. During follow-up, amounts of serum acylcarnitines and urinary organic acids fluctuated paralleling serum transaminases, with highest values manifest during periods of illness (e.g., flu) or catabolic stress (e.g., protracted fasting), confirming the recurrent nature of episodes in MADD (Supplementary Table 1 and Figures 2A,B).
After informed consent from the parents, a genetic test to confirm the diagnosis and to identify the carrier subjects in the family was therefore requested. Molecular analysis of genomic DNA isolated from peripheral whole blood was performed by Sanger sequencing of coding regions, intron/exon boundaries and 5 ′ and 3 ′ UTR of ETFA, ETFB, and ETFDH genes, using previously described protocol (5). Genetic results revealed the presence of the novel missense variant c.1658A > G (p.Tyr553Cys) in exon 12 of the ETFDH gene in the patient, at homozygous state. Both parents were heterozygous for the mutated allele. The proband's sister did not show the variant ( Figure 1E).
Lipid restricted diet and Riboflavin treatment (100 mg thrice/day) ameliorated dramatically the asthenic symptoms. Repeat 6 min walking test showed no/minimal fatigue after 2 months (oxygen saturation 100%, heart frequency 150 beats per minute, 560 m traveled). Liver brightness at ultrasonography, liver function tests, along with the whole hepatic, muscular and metabolic laboratory profiles improved remarkably and persistently (Supplementary Table 1). Control Magnetic Resonance Imaging, after 10 months of therapy, showed absent/sharp decrease of lipid storage of thighs muscles ( Figure 1D).

LITERATURE REVIEW
We searched within the PubMed, Scopus, and Cochrane Library academic medical databases. The database search strategy was formulated around terms for MADD AND several other text words reported in Supplementary Table 2 To be eligible for inclusion, studies had to describe a case of MADD associated with liver disease.
Study details and quality characteristics were independently extracted by three of the authors for all the articles and in a stepwise approach, first by reading the title, then by reviewing the abstract, and finally by revising the full text, where appropriate ( Supplementary Figure 1). At the end of revision, 37 studies were selected.
Out of 270 reported patients, 70 had liver involvement. Fatty liver (45 cases) and recurrent vomiting represents rare extramuscular symptoms of late onset MADD, and it has been describsed especially in mainland Chinese patients (3). Table 1 shows literature cases reported with fatty liver and/or liver disease as compared with our patient. The ages of the patients included ranged from six months to 68 years at diagnosis. Only 9 out of the 45 patients had severe fatty liver either at imaging or at liver histology. One patient showed early stages of liver failure, one recurrent pancreatitis, and one recurrent rhabdomyolysis and acute renal failure after the age of 46.

DISCUSSION
Differently from adult age, where the causes of hypertransaminasemia and/or fatty liver other than obesityrelated liver disease are limited, the pediatric diagnostic approach may be more complex. In addition to nonalcoholic fatty liver disease (NAFLD)/metabolic dysfunction-associated fatty liver disease (MAFLD) children, in fact, may present individually rare genetic/metabolic conditions which collectively end up into a relevant and challenging ≥30% group of heterogeneous pediatric onset liver diseases (42)(43)(44). These, therefore, still require a cautious exclusion, so that pediatric fatty liver disease (PeFLD) with some subtypes has been proposed to be a better terminology to use and to be subject to revisitation upon reaching adulthood (45). In our patient urinary organic acids, serum amino acids and acyl carnitine patterns were crucial to suggest the diagnosis and pave the way to the subsequent confirmatory tests of a late onset MADD-related fatty liver disease. The latter is a condition which is more frequently characterized by prevalent metabolic and muscular signs rather than by severe fatty liver as confirmed by our systematic review of the literature ( Table 1). Molecular analysis of ETFA, ETFB, and ETFDH genes indicated that our patient was homozygous for the missense variant c.1658 A>G (p.Tyr553Cys) in exon 12 of the ETFDH gene. This variant is absent in the public database of mutations and polymorphisms. Interestingly, in the literature a pathogenic missense mutation (c.1657T > C) is reported in the same codon, determining a different amino acid substitution (p.Tyr553His). Including this observation in the pathogenetic evaluation, performed according to the American College of Medical Genetics and Genomics (ACMG) guidelines for variant interpretation (38), the novel variant c.1657T > C (p.Tyr553Cys) is classifiable as likely pathogenetic. Therefore, we identified a novel missense mutation in the ETFDH gene associated to late-onset riboflavin-responsive MADD.
The clinical phenotype of type III MADD is highly variable and ranges from acute, in some cases even fatal, metabolic crises in infancy to asymptomatic adults. This intriguing variability may probably be explained by the observation that even minute amounts of residual ETF/ETFDH activity seem to be sufficient to prevent embryonic development of congenital anomalies. Studies of an Asp128Asn mutation of the ETFB gene, identified in a patient with type III disease, showed that the residual activity of the enzyme could be rescued up to 59% of that of wild type activity when ETFB-(Asp128Asn)-transformed E. coli cells were grown at low temperature. This suggests that the environmental factors such as cellular temperature and stress may influence the enzymatic phenotype (46) in agreement with the case history of our patient. Although most patients become symptomatic within the first two decades, onset of symptoms ranges from the second month of life to late adulthood. Decompensations are characterized by acidosis, hypoglycemia, elevated activities of transaminases, rhabdomyolysis with raised creatine kinase activity, and, eventually, hyperammonemia. These episodes are usually triggered by catabolic states, either due to infections and febrile illnesses or to a reduced energy supply (1). In our case, the clinical presentation was prevalently characterized by some vomiting episodes and fatigue and recurrent severe hypertransaminasemia with a histological correlate of massive micro-macrovacuolar steatohepatitis with minimal fibrosis. Lipid restricted diet and riboflavin treatment dramatically ameliorated the clinical symptoms, liver brightness and LFTs, and metabolic profiles as reported in literature for the late onset MADD, which is therefore also called riboflavin-responsive MADD (RR-MADD) (3,47). Riboflavin, the precursor of the coenzyme FAD, acts as a molecular chaperone that promotes folding and steady state levels of misfolded ETF-QO proteins in early stages and stabilizes folding intermediates or membrane-inserted proteins in later stages (48).

CONCLUSIONS
RR-MADD is a treatable but rare disease and its diagnosis is difficult due to its high clinical phenotypic heterogeneity (13). Prevalent hepatic disease rather than isolated lipidic myopathy is a much less common presentation of the lateonset form. Our study is significant as it suggests that one should have consideration for this condition in the workup of an otherwise orphan diagnosis of obesity-unrelated severe steatohepatitis with recurrent hypertransaminasemia. Correct diagnosis of MADD type III may allow timely initiation of a sometimes lifesaving riboflavin treatment and improve outcome.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

ETHICS STATEMENT
Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin. Written informed consent was obtained from the minor(s)' legal guardian/next of kin for the publication of any potentially identifiable images or data included in this article.

AUTHOR CONTRIBUTIONS
MAS and CM conceived the original idea and took the lead in writing the manuscript with input from all authors. LN, GI, and GPC followed the clinical course of the patient over time. CB made the histopathology study. ST and FDS made MRI studies. FB, CM, MR, and GF carried out biochemical and molecular studies. DM and PV gave substantial intellectual contribution. PV is the guarantor of the study. All authors provided critical feedback and helped shape the study, discussed the results, and contributed to the final manuscript.