Variations in IBD (ACAD8) in children with elevated C4-carnitine detected by tandem mass spectrometry newborn screening.

The isobutyryl-CoA dehydrogenase (IBD) enzyme is involved in the degradation of valine. IBD deficiency was first reported in 1998 and subsequent genetic investigations identified acyl-CoA dehydrogenase (ACAD) 8, now IBD, as the gene responsible for IBD deficiency. Only three individuals homozygous or compound heterozygous for variations in the IBD gene have been reported. We present IBD deficiency in an additional four newborns with elevated C(4)-carnitine identified by tandem mass spectrometry (MS/MS) screening in Denmark and the United States. Three showed urinary excretions of isobutyryl-glycine, and in vitro probe analysis of fibroblasts from two newborns indicated enzymatic IBD defect. Molecular genetic analysis revealed seven new rare variations in the IBD gene (c.348C>A, c.400G>T, c.409G>A, c.455T>C, c.958G>A, c.1000C>T and c.1154G>A). Furthermore, sequence analysis of the short-chain acyl-CoA dehydrogenase (SCAD) gene revealed heterozygosity for the prevalent c.625G>A susceptibility variation in all newborns and in the first reported IBD patient. Functional studies in isolated mitochondria demonstrated that the IBD variations present in the Danish newborn (c.409G>A and c.958G>A) together with a previously published IBD variation (c.905G>A) disturbed protein folding and reduced the levels of correctly folded IBD tetramers. Accordingly, low/no IBD residual enzyme activity was detectable when the variant IBD proteins were overexpressed in Chang cells.

Short/branched-chain acyl-CoA dehydrogenase deficiency due to an IVS3+3A>G mutation that causes exon skipping.

Short/branched-chain acyl-CoA dehydrogenase deficiency (SBCADD) is an autosomal recessive disorder of L: -isoleucine catabolism. Little is known about the clinical presentation associated with this enzyme defect, as it has been reported in only a limited number of patients. Because the presence of C5-carnitine in blood may indicate SBCADD, the disorder may be detected by MS/MS-based routine newborn screening. It is, therefore, important to gain more knowledge about the clinical presentation and the mutational spectrum of SBCADD. In the present study, we have studied two unrelated families with SBCADD, both with seizures and psychomotor delay as the main clinical features. One family illustrates the fact that affected individuals may also remain asymptomatic. In addition, the normal level of newborn blood spot C5-acylcarnitine in one patient underscores the fact that newborn screening by MS/MS currently lacks sensitivity in detecting SBCADD. Until now, seven mutations in the SBCAD gene have been reported, but only three have been tested experimentally. Here, we identify and characterize an IVS3+3A>G mutation (c.303+3A>G) in the SBCAD gene, and provide evidence that this mutation is disease-causing in both families. Using a minigene approach, we show that the IVS3+3A>G mutation causes exon 3 skipping, despite the fact that it does not appear to disrupt the consensus sequence of the 5' splice site. Based on these results and numerous literature examples, we suggest that this type of mutation (IVS+3A>G) induces missplicing only when in the context of non-consensus (weak) 5' splice sites. Statistical analysis of the sequences shows that the wild-type versions of 5' splice sites in which +3A>G mutations cause exon skipping and disease are weaker on average than a random set of 5' splice sites. This finding is relevant to the interpretation of the functional consequences of this type of mutation in other disease genes.

DNA-based prenatal diagnosis for severe and variant forms of multiple acyl-CoA dehydrogenation deficiency.

OBJECTIVES: Multiple acyl-CoA dehydrogenation deficiency (MADD) is a clinically heterogeneous disorder of mitochondrial fatty acid, amino acid, and choline oxidation due to mutations in the genes encoding electron transfer flavoprotein (ETF) or ETF ubiquinone oxidoreductase (ETFQO). So far, prenatal diagnosis of MADD has relied mostly on second-trimester biochemical analyses of amniotic fluid or cultured amniocytes. We report here on an alternative DNA-based approach for prenatal diagnosis in pregnancies at risk of MADD. METHODS: We used our knowledge of the mutational status in three unrelated families with a history of MADD to perform direct sequencing for the familial mutations using genomic DNA isolated from chorionic villus samples (CVS) at gestational week 10 to 11. RESULTS: Within two days, we were able to carry out accurate DNA-based prenatal testing in one pregnancy at risk of severe MADD, and in two pregnancies at risk of variant forms of MADD. CONCLUSION: This is the first report of DNA-based prenatal diagnosis of MADD. Our molecular approach is suitable for fast and reliable first-trimester prenatal diagnosis in pregnancies at risk of severe and variant forms of MADD.