169 6 iPSC-derived liver organoids as a tool to study Medium Chain Acyl-CoA Dehydrogenase deficienc INTRODUCTION Medium-chain acyl-CoA dehydrogenase (MCAD) is one of the flavoenzymes that catalyze the first step of mitochondrial β-oxidation of fatty acids. It oxidizes medium-chain acyl-CoA into 2-enoyl-CoA. MCAD deficiency (MCADD) is an autosomal recessive disease and the most common fatty acid oxidation disorder (FAOD) with a prevalence of 1/8,300 in the Netherlands1. Interestingly, people homozygous for an ACADM mutation can present various clinical phenotypes. While some patients remain asymptomatic throughout life, some experience life-threatening hypoketotic hypoglycemia when exposed to catabolic stress (e.g. fasting and intercurrent illness)2–4. After diagnosis, the most common treatment relies on avoidance of fasting and an emergency regimen5,6. While the mutation c.985A>G (p.K329E) is estimated to account for more than 90% of pathogenic alleles, the implementation of MCADD in newborn screening has revealed several ACADM variants of unknown significance7. The clinical risk associated with individual variants and the mechanisms underlying metabolic decompensation remain unclear7,8. Patients with residual MCAD activity, equal or above 10%, have been referred to as mild and often remain asymptomatic1. However, within the group that is homozygous for the classical c.985A>G (p.K329E) mutation, patients may present with a wide range of symptoms and disease severity, suggesting a limited genotype-phenotype correlation. Therefore, it is likely that genetic variation beyond the ACADM gene, the environment, and epigenetics play a major role in the development of a metabolic crisis9. Preclinical, mechanistic studies of the pathophysiology underlying MCADD have traditionally relied on the use of animal models10–12. However, rodents are equipped with an extra dehydrogenase, long-chain acyl-CoA dehydrogenase (LCAD), which shows overlapping substrate specificity with MCAD13 and may therefore mask the phenotype. Additionally, a full ACADM deletion cannot elucidate the effect of individual point mutations in the ACADM gene. This makes human and patient-derived in vitro models an attractive alternative for the study of MCADD. Organoids are 3D multicellular structures that proliferate in vitro, while recapitulating several functions of the organ of origin. They can be obtained from primary tissue as well as from pluripotent and adult stem cells14. Liver organoids recapitulate several hepatic functions such as albumin production, bile acid production, and CYP3A4 activity15. Since they were first described16, organoids have been used for an increasing number of applications including the study of differentiation, organ development17, and (metabolic) disease18, as well as drug screening 19. For primary tissue, highly invasive liver
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