Multiple acyl-CoA dehydrogenation deficiency (MADD) is a disorder of fatty acid, amino acid and choline metabolism that can result from defects in two flavoproteins, electron transfer flavoprotein (ETF) or ETF: ubiquinone oxidoreductase (ETF:QO). Some patients respond to pharmacological doses of riboflavin. It is unknown whether these patients have defects in the flavoproteins themselves or defects in the formation of the cofactor, FAD, from riboflavin. We report 15 patients from 11 pedigrees. All the index cases presented with encephalopathy or muscle weakness or a combination of these symptoms; several had previously suffered cyclical vomiting. Urine organic acid and plasma acyl-carnitine profiles indicated MADD. Clinical and biochemical parameters were either totally or partly corrected after riboflavin treatment. All patients had mutations in the gene for ETF:QO. In one patient, we show that the ETF:QO mutations are associated with a riboflavin-sensitive impairment of ETF:QO activity. This patient also had partial deficiencies of flavin-dependent acyl-CoA dehydrogenases and respiratory chain complexes, most of which were restored to control levels after riboflavin treatment. Low activities of mitochondrial flavoproteins or respiratory chain complexes have been reported previously in two of our patients with ETF:QO mutations. We postulate that riboflavin-responsive MADD may result from defects of ETF:QO combined with general mitochondrial dysfunction. This is the largest collection of riboflavin-responsive MADD patients ever reported, and the first demonstration of the molecular genetic basis for the disorder.

Precursor mRNA splicing homeostasis is a biomarker and predictor of life expectancy in Caenorhabditis elegans and defects in global pre-mRNA splicing associated with age are reduced by dietary restriction via splicing factor 1. The collapse of protein homeostasis and increasing error rates in transcription are key risk factors for various chronic diseases, and are associated with ageing. William Mair and colleagues demonstrate that pre-mRNA splicing homeostasis is a biomarker and predictor of life expectancy in Caenorhabditis elegans, and that defects in global pre-mRNA splicing associated with age are reduced by dietary restriction via splicing factor 1 (SFA-1). SFA-1 is specifically required for lifespan extension both by dietary restriction, and modulation of TORC1 pathway components. Overexpression of SFA-1 is sufficient to extend lifespan. This work suggests that modulation of specific spliceosome components can promote healthy ageing. Ageing is driven by a loss of transcriptional and protein homeostasis1,2,3 and is the key risk factor for multiple chronic diseases. Interventions that attenuate or reverse systemic dysfunction associated with age therefore have the potential to reduce overall disease risk in the elderly. Precursor mRNA (pre-mRNA) splicing is a fundamental link between gene expression and the proteome, and deregulation of the splicing machinery is linked to several age-related chronic illnesses4,5. However, the role of splicing homeostasis in healthy ageing remains unclear. Here we demonstrate that pre-mRNA splicing homeostasis is a biomarker and predictor of life expectancy in Caenorhabditis elegans. Using transcriptomics and in-depth splicing analysis in young and old animals fed ad libitum or subjected to dietary restriction, we find defects in global pre-mRNA splicing with age that are reduced by dietary restriction via splicing factor 1 (SFA-1; the C. elegans homologue of SF1, also known as branchpoint binding protein, BBP). We show that SFA-1 is specifically required for lifespan extension by dietary restriction and by modulation of the TORC1 pathway components AMPK, RAGA-1 and RSKS-1/S6 kinase. We also demonstrate that overexpression of SFA-1 is sufficient to extend lifespan. Together, these data demonstrate a role for RNA splicing homeostasis in dietary restriction longevity and suggest that modulation of specific spliceosome components may prolong healthy ageing.

CUGBP1 and MBNL1 are developmentally regulated RNA-binding proteins that are causally associated with myotonic dystrophy type 1. We globally determined the in vivo RNA-binding sites of CUGBP1 and MBNL1. Interestingly, CUGBP1 and MBNL1 are both preferentially bound to 3′ UTRs. Analysis of CUGBP1- and MBNL1-bound 3′ UTRs demonstrated that both factors mediate accelerated mRNA decay and temporal profiles of expression arrays supported this. Role of CUGBP1 on accelerated mRNA decay has been previously reported, but the similar function of MBNL1 has not been reported to date. It is well established that CUGBP1 and MBNL1 regulate alternative splicing. Screening by exon array and validation by RT-PCR revealed position dependence of CUGBP1- and MBNL1-binding sites on the resulting alternative splicing pattern. This study suggests that regulation of CUGBP1 and MBNL1 is essential for accurate control of destabilization of a broad spectrum of mRNAs as well as of alternative splicing events.

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. Hum Mutat 22:12–23, 2003.