Molecular and functional characterisation of mild MCAD deficiencyZschocke, J.; Schulze, A.; Lindner, M.; Fiesel, S.; Olgemöller, K.; Hoffmann, G.F.; Penzien, J.; Ruiter, J.P.N.; Wanders, R.J.A.; Mayatepek, E. General rights It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulationsIf you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: http://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. Download date: 08 May 2018Abstract We report a novel mild variant of medium-chain acyl-CoA dehydrogenase deficiency (MCADD) diagnosed in four infants who, in neonatal screening, showed abnormal acylcarnitine profiles indicative of MCADD. Three patients showed completely normal urinary organic acids and phenylpropionic acid loading tests were normal in all four patients. Enzyme studies showed residual MCAD activities between "classical" MCADD and heterozygotes. ACADM gene analysis revealed compound heterozygosity for the common mutation K329E and a novel mutation, Y67H, in two cases, and homozygosity for mutation G267R and the novel mutation S245L, respectively, in two children of consanguineous parents. As in other metabolic disorders, the distinction between "normal" and "disease" in MCAD deficiency is blurring into a spectrum of enzyme deficiency states caused by different mutations in the ACADM gene potentially influenced by factors affecting intracellular protein processing.

Deficiency of mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (mHMGS) is a recessive disorder of ketogenesis that has been previously diagnosed in two children with hypoglycaemic hypoketotic coma during fasting periods. Here, we report the results of molecular investigations in a third patient affected by this disease. Sequencing of the entire coding region of the HMGCS2 gene revealed two missense mutations, G212R and R500H. Mendelian inheritance was confirmed by the analysis of parental samples and neither of the mutations was found on 200 control chromosomes. Functional relevance was confirmed by in vitro expression studies in cytosolic HMGS-deficient cells. Whereas wild-type cDNA of the HMGCS2 gene reverted the auxotrophy for mevalonate, the cDNAs of the mutants did not. The disease may be recognised by specific clinical and biochemical features but it is difficult to confirm enzymatically since the gene is expressed only in liver and testis. Molecular studies may facilitate or confirm future diagnoses in affected patients.

Transient gene expression (TGE) in HEK293 cells was optimized by Vink et al. byco-expression of human cell cycle inhibitors p21 CIP /p27 KIP and Simian virus 40 large T antigen (SVLT). In this study, we investigated the effect of this enhancer protein complex on the TGE experiments in a cell-cycle arrested condition of HEK293F cells induced by valproic acid. Growth profiles, consumptions of nutrients, formations of waste products, and product titers of recombinant human antibodies (huAb) were monitored during the 7-day cultivation time. Our results showed that the use of enhancer proteins increased the product yields in a growth arrest condition as well. During the growth phase, no differences were detected regarding viable cell densities (VCDs), viabilities, growth rates, and cell diameters between the TGE experiments with and without enhancer proteins. However, during the declining phase VCD and viability showed slightly higher values at day 6 and 7 in the presence of enhancers. Furthermore, we could not detect any differences in glucose and glutamine metabolism during batch cultivations with co-expression of enhancer proteins. Taken together, the special complex of enhancer proteins did not contribute to further enhancement of growth arrest and shift in the main cell metabolisms, but resulted in higher cell viability during the decline phase. Our observations suggest that the human cell cycle inhibitors p21 CIP /p27 KIP together with very low amount of SVLT antigen may induce alternative functional activities than growth arrest to further improve the yield of recombinant proteins. K E Y W O R D S antibody production, cell cycle inhibitors, HEK293, SV40 large T antigen, transient gene expression 1 | INTRODUCTION In the process of antibody engineering and high-throughput screening, a simple, rapid, and efficient method of recombinant protein expression is required. Transient gene expression (TGE) in eukaryotic cells is a wellestablished method and has an advantage over stable expression since it has shorter timelines for protein production. 1,2 In the biotechnology industry, the most commonly used mammalian hosts for the production of recombinant proteins are human embryonic kidney (HEK) 293 cells 3 and Chinese hamster ovary (CHO) cells. 4,5 HEK293 cells provide an advantage in the expression of human recombinant proteins due to the pattern of their post-translational modifications. 6 Structure, number and location of post-translational N-glycans are known to affect the biologic activity, stability and half-life of recombinant proteins. 7 To enable cost-effective and efficient high yielding transient transfections, several strategies have been developed. Most of these