A Novel Tandem Mass Spectrometry Method for Rapid Confirmation of Medium- and Very Long-Chain acyl-CoA Dehydrogenase Deficiency in Newborns

BACKGROUND:Newborn screening for medium- and very long-chain acyl-CoA dehydrogenase (MCAD and VLCAD, respectively) deficiency, using acylcarnitine profiling with tandem mass spectrometry, has increased the number of patients with fatty acid oxidation disorders due to the identification of additional milder, and so far silent, phenotypes. However, especially for VLCADD, the acylcarnitine profile can not constitute the sole parameter in order to reliably confirm disease. Therefore, we developed a new liquid chromatography tandem mass spectrometry (LC-MS/MS) method to rapidly determine both MCAD- and/or VLCAD-activity in human lymphocytes in order to confirm diagnosis. METHODOLOGY:LC-MS/MS was used to measure MCAD- or VLCAD-catalyzed production of enoyl-CoA and hydroxyacyl-CoA, in human lymphocytes. PRINCIPAL FINDINGS:VLCAD activity in controls was 6.95+/-0.42 mU/mg (range 1.95 to 11.91 mU/mg). Residual VLCAD activity of 4 patients with confirmed VLCAD-deficiency was between 0.3 and 1.1%. Heterozygous ACADVL mutation carriers showed residual VLCAD activities of 23.7 to 54.2%. MCAD activity in controls was 2.38+/-0.18 mU/mg. In total, 28 patients with suspected MCAD-deficiency were assayed. Nearly all patients with residual MCAD activities below 2.5% were homozygous 985A>G carriers. MCAD-deficient patients with one other than the 985A>G mutation had higher MCAD residual activities, ranging from 5.7 to 13.9%. All patients with the 199T>C mutation had residual activities above 10%. CONCLUSIONS:Our newly developed LC-MS/MS method is able to provide ample sensitivity to correctly and rapidly determine MCAD and VLCAD residual activity in human lymphocytes. Importantly, based on measured MCAD residual activities in correlation with genotype, new insights were obtained on the expected clinical phenotype

Footprint-free human fetal foreskin derived iPSCs: A tool for modeling hepatogenesis associated gene regulatory networks

Induced pluripotent stem cells (iPSCs) are similar to embryonic stem cells and can be generated from somatic cells. We have generated episomal plasmid-based and integration-free iPSCs (E-iPSCs) from human fetal foreskin fibroblast cells (HFF1). We used an E-iPSC-line to model hepatogenesis in vitro. The HLCs were characterized biochemically, i.e. glycogen storage, ICG uptake and release, UREA and bile acid production, as well as CYP3A4 activity. Ultra-structure analysis by electron microscopy revealed the presence of lipid and glycogen storage, tight junctions and bile canaliculi- all typical features of hepatocytes. Furthermore, the transcriptome of undifferentiated E-iPSC, DE, HE and HLCs were compared to that of fetal liver and primary human hepatocytes (PHH). K-means clustering identified 100 clusters which include developmental stage-specific groups of genes, e.g. OCT4 expression at the undifferentiated stage, SOX17 marking the DE stage, DLK and HNF6 the HE stage, HNF4α and Albumin is specific to HLCs, fetal liver and adult liver (PHH) stage. We use E-iPSCs for modeling gene regulatory networks associated with human hepatogenesis and gastrulation in general

Functional Effects of Different Medium-Chain Acyl-CoA Dehydrogenase Genotypes and Identification of Asymptomatic Variants

<div><p>Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency (OMIM 201450) is the most common inherited disorder of fatty acid metabolism presenting with hypoglycaemia, hepatopathy and Reye-like symptoms during catabolism. In the past, the majority of patients carried the prevalent c.985A>G mutation in the <em>ACADM</em> gene. Since the introduction of newborn screening many other mutations with unknown clinical relevance have been identified in asymptomatic newborns. In order to identify functional effects of these mutant genotypes we correlated residual MCAD (OMIM 607008) activities as measured by octanoyl-CoA oxidation in lymphocytes with both genotype and relevant medical reports in 65 newborns harbouring mutant alleles. We identified true disease-causing mutations with residual activities of 0 to 20%. In individuals carrying the c.199T>C or c.127G>A mutation on one allele, residual activities were much higher and in the range of heterozygotes (31%–60%). Therefore, both mutations cannot clearly be associated with a clinical phenotype. This demonstrates a correlation between the octanoyl-CoA oxidation rate in lymphocytes and the clinical outcome. With newborn screening, the natural course of disease is difficult to assess. The octanoyl-CoA oxidation rate, therefore, allows a risk assessment at birth and the identification of new <em>ACADM</em> genotypes associated with asymptomatic disease variants.</p> </div

Fatal neonatal encephalopathy and lactic acidosis caused by a homozygous loss-of-function variant in <em>COQ9</em>.

Coenzyme Q10 (CoQ10) has an important role in mitochondrial energy metabolism by way of its functioning as an electron carrier in the respiratory chain. Genetic defects disrupting the endogenous biosynthesis pathway of CoQ10 may lead to severe metabolic disorders with onset in early childhood. Using exome sequencing in a child with fatal neonatal lactic acidosis and encephalopathy, we identified a homozygous loss-of-function variant in COQ9. Functional studies in patient fibroblasts showed that the absence of the COQ9 protein was concomitant with a strong reduction of COQ7, leading to a significant accumulation of the substrate of COQ7, 6-demethoxy ubiquinone10. At the same time, the total amount of CoQ10 was severely reduced, which was reflected in a significant decrease of mitochondrial respiratory chain succinate-cytochrome c oxidoreductase (complex II/III) activity. Lentiviral expression of COQ9 restored all these parameters, confirming the causal role of the variant. Our report on the second COQ9 patient expands the clinical spectrum associated with COQ9 variants, indicating the importance of COQ9 already during prenatal development. Moreover, the rescue of cellular CoQ10 levels and respiratory chain complex activities by CoQ10 supplementation points to the importance of an early diagnosis and immediate treatment

Genotype and octanoyl-CoA oxidation measured in lymphocytes of 65 subjects with suspected MCAD deficiency^a.

a<p>Activity was given as % of the mean value of healthy controls (1.89±0.58 mU/mg).</p>b<p>Newborn screening octanoyl-carnitine results were given in µmol/L. The cut-off values were given bracketed.</p>c<p>Data not available.</p>d<p>no HPLC-UV measurement performed.</p>e<p>novel mutation.</p