Mutations in electron transfer flavoprotein (ETF) and its dehydrogenase (ETFDH) are the molecular basis of multiple acyl-CoA dehydrogenation deficiency (MADD), an autosomal recessively inherited and clinically heterogeneous disease that has been divided into three clinical forms: a neonatal-onset form with congenital anomalies (type I), a neonatal-onset form without congenital anomalies (type II), and a late-onset form (type III). To examine whether these different clinical forms could be explained by different ETF/ETFDH mutations that result in different levels of residual ETF/ETFDH enzyme activity, we have investigated the molecular genetic basis for disease development in nine patients representing the phenotypic spectrum of MADD. We report the genomic structures of the ETFA, ETFB, and ETFDH genes and the identification and characterization of seven novel and three previously reported disease-causing mutations. Our molecular genetic investigations of these nine patients are consistent with three clinical forms of MADD showing a clear relationship between the nature of the mutations and the severity of disease. Interestingly, our data suggest that homozygosity for two null mutations causes fetal development of congenital anomalies resulting in a type I disease phenotype. Even minute amounts of residual ETF/ETFDH activity seem to be sufficient to prevent embryonic development of congenital anomalies giving rise to type II disease. Overexpression studies of an ETFB-D128N missense mutation identified in a patient with type III disease showed that the residual activity of the mutant enzyme could be rescued up to 59% of that of wild-type activity when ETFB-D128N-transformed E. coli cells were grown at low temperature. This indicates that the effect of the ETF/ETFDH genotype in patients with milder forms of MADD, in whom residual enzyme activity allows modulation of the enzymatic phenotype, may be influenced by environmental factors like cellular temperature.

Clear relationship between ETF/ETFDH genotype and phenotype in patients with multiple acyl-CoA dehydrogenation deficiency.

A Lactobacillus brevis strain with the ability to synthesize butanol from glucose was constructed by metabolic engineering. The genes crt, bcd, etfB, etfA, and hbd, composing the bcs-operon, and the thl gene encode the enzymes of the lower part of the clostridial butanol pathway (crotonase, butyryl-CoA-dehydrogenase, two subunits of the electron transfer flavoprotein, 3-hydroxybutyryl-CoA dehydrogenase, and thiolase) of Clostridium acetobutylicum. They were cloned into the Gram-positive/Gram-negative shuttle plasmid vector pHYc. The two resulting plasmids pHYc-thl-bcs and pHYc-bcs (respectively, with and without the clostridial thl gene) were transferred to Escherichia coli and L. brevis. The recombinant L. brevis strains were able to synthesize up to 300 mg l−1 or 4.1 mM of butanol on a glucose-containing medium. A L. brevis strain carrying the clostridial bcs-operon has the ability to synthesize butanol with participation of its own thiolase, aldehyde dehydrogenase, and alcohol dehydrogenase. The particular role of the enzymes involved in butanol production and the suitability of L. brevis as an n-butanol producer are discussed.

Reconstructing the clostridial n-butanol metabolic pathway in Lactobacillus brevis

To date, seven inborn errors of mitochondrial fatty acid oxidation have been identified. A total of about 100 patients in the world have been reported. Clinically the β-oxidation defects are more often characterized by episodic hypoglycaemia leading to a coma mimicking Reye's syndrome. The hypoglycaemia is non-ketotic since the synthesis of ketone bodies is deficient. Periods of decompensation occur when carbohydrate supply is poor, e.g. prolonged fasting, vomiting, or increased caloric requirements, as and when lipid stores are used. Defects in β-oxidation have also been reported to be one cause of sudden infant death syndrome. The diagnosis of these inborn errors is by biochemical investigation since where symptoms suggest such a defect, the precise aetiology cannot be assessed. The biochemical diagnosis is based firstly on identification of abnormal plasma and of urinary metabolites during acute attacks. Derivatives of the ω-oxidation and ω-1-oxidation of medium chain fatty acids have been identified, as well as acylglycine and acylcarnitine conjugates. These metabolites are nearly always absent when patients are in good clinical condition. Secondly, the diagnosis must be based on the identification of the enzymatic defects: this involves global assays which allow a localization of the ‘level’ of the defect (i.e. the oxidation of long, medium or short chain fatty acids) and specific measurement of enzyme activities (acyl-CoA dehydrogenases and electron carriers: ETF and ETF-DH). The diagnosis of these disorders is of prime importance because of the severity of the clinical symptoms. These can be prevented, in some cases, by an appropriate diet (a high carbohydrate, low fat diet, sometimes supplemented withl-carnitine). In other cases, genetic counselling can be offered.

The inborn errors of mitochondrial fatty acid oxidation.

Mitochondrial fatty acid oxidation defects have been recognized since the early 1970s. The discovery rate has been rather constant, with 3–4 ‘new’ disorders identified every decade and with the most recent example, ACAD9 deficiency, reported in 2007. In this presentation we will focus on three of the ‘old’ defects: medium-chain acyl-CoA dehydrogenase (MCAD) deficiency, riboflavin responsive multiple acyl-CoA dehydrogenation (RR-MAD) deficiency, and short-chain acyl-CoA dehydrogenase (SCAD) deficiency. These disorders have been discussed in many publications and at countless conference presentations, and many questions relating to them have been answered. However, continuing clinical and pathophysiological research has raised many further questions, and new ideas and methodologies may be required to answer these. We will discuss these challenges. For MCAD deficiency the key question is why 80% of symptomatic patients are homozygous for the prevalent ACADM gene variation c.985A > G whereas this is found in only ∼50% of newborns with a positive screen. For RR-MAD deficiency, the challenge is to find the connection between variations in the ETFDH gene and the observed deficiency of a number of different mitochondrial dehydrogenases as well as deficiency of FAD and coenzyme Q10. With SCAD deficiency, the challenge is to elucidate whether ACADS gene variations are disease-associated, especially when combined with other genetic/cellular/environmental factors, which may act synergistically.

Mitochondrial fatty acid oxidation defects—remaining challenges

Objective.– To investigate the influence of different pharmacological treatments on the intensity dependence of auditory evoked cortical potentials in migraineurs.



Background.– Between attacks, patients with migraine show abnormalities in cortical information processing and decreased brain mitochondrial energy reserve. Both are most probably relevant for migraine pathogenesis, and they could be differentially modified by prophylactic drug therapy.



Design.–The intensity dependence of the auditory evoked cortical potentials is, on average, increased in migraine. We have studied this intensity dependence in 26 patients before and after a 4-month period of prophylaxis with beta-blockers (n=11, all migraine without aura; metoprolol or bisoprolol) or riboflavin (n=15, migraine without aura: 13, migraine with aura: 2). Recordings were performed at least 3 days before or after an attack.



Results.– After the treatment with beta-blockers, the intensity dependence of the auditory evoked cortical potentials was significantly decreased (before: 1.66 ± 1.02 μV/10 dB; after: 0.79 ± 1.06 μV/10 dB, P=.02). The decrease in intensity dependence was correlated significantly with clinical improvement (r=.69, P=.02). There was no change in intensity dependence after riboflavin treatment (before: 1.80 ± 0.81 μV/10 dB; after: 1.56 ± 0.83 μV/10 dB, P=.39), although the majority of patients showed improvement.



Conclusions.– These results confirm that beta-blockers and riboflavin act on two distinct pathophysiological mechanisms. Combining both treatments might enhance their efficacy without increasing central nervous system side effects.

Prophylactic treatment of migraine with beta-blockers and riboflavin: differential effects on the intensity dependence of auditory evoked cortical potentials.

The key reaction in the β-oxidation of fatty acids is the acyl-CoA dehydrogenation, catalyzed by short chain, medium chain, and long chain acyl-CoA dehydrogenases. Acyl-CoA dehydrogenation reactions are also involved in the metabolism of the branched chain amino acids, where isovaleryl-CoA and 2-methylbutyryl-CoA dehydrogenases are involved and in the metabolism of lysine, 5-hydroxylysine and tryptophan, where glutaryl-CoA dehydrogenase functions. In all of these dehydrogenation systems reducing equivalents are transported to the main respiratory chain by electron transfer flavoprotein (ETF) and electron transfer flavoprotein dehydrogenase (ETFDH), which are common to all the dehydrogenation systems. The acyl-CoA dehydrogenation enzymes are dependent on flavin adenine dinucleotide (FAD) as coenzyme, for which riboflavin is the precursor. Patients with multiple acyl-CoA dehydrogenation deficiencies have been found in whom the defect has been located to ETF and/or ETFDH. A few patients with multiple acyl-CoA dehydrogenation deficiencies have been described, in whom no defects in acyl-CoA dehydrogenases, ETF or ETFDH have been found but who respond clinically and biochemically to pharmacological doses of riboflavin. This indicates a defect related to the metabolism of FAD. An uptake defect of riboflavin or a synthesis defect of FAD from riboflavin have been excluded byin vivo andin vitro studies. A mitochondrial transport defect of FAD or a defect in the binding FAD to ETF and/or ETFDH remains possible.

N. Gregersen

Papers

Riboflavin-responsive Defects of β-Oxidation