Metabolische Charakterisierung einer neuartigen humanen Mutation von APOA5

Abstract

APOA5 ist einer der Hauptregulatoren des murinen und humanen Stoffwechsels und vermittelt die Lipoproteinaufnahme in periphere Gewebe. APOA5-defiziente Mäuse zeigen eine starke Hypertriglyzeridämie mit 10-50-fach erhöhten Werten. Diese Dissertation beschreibt eine neuartige humane Mutation von APOA5 in einem 11-Monate alten Säugling, welche für einen Verlust von acht Aminosäuren innerhalb der kodierenden Region des Signal Peptids verantwortlich ist. Diese Mutation führt zu einem Verlust des hydrophoben Kerns des Signal Peptids, sodass es zu einem Funktionsverlust und einer intrazellulären Fehlsortierung des Proteins kommt. Diese Mutation führte im Patienten zu einer Hypertriglyzeridämie und einer Pankreatitis. Therapeutisch konnte durch Abstillen eine Reduktion der Triglyzeride auf fast Normwerte erreicht werden, sodass der Patient wahrscheinlich auf eine lebenslange fettarme Ernährung angewiesen ist, um rezidivierende Pankreatitiden zu vermeiden.

APOA5 is a key player in murine and human metabolism as it mediates – at least in part – disposal and retention of lipoproteins. APOA5-deficient mice display a tremendous increase in plasma lipids, with increased amounts of triglycerides of more than 10- 50 fold of healthy wild-type reference cohorts. It is also known, that under certain condi-tions, VLDL particle synthesis is influenced by murine Apoa5 as well, leading to high plasma triglycerides in deficient mice. For humans, no clear VLDL synthesis effect could be shown, but the VLDL catabolism was markedly decreased, which is likely to indicate the blood phenotype. APOA5 is able to increase the activity of the lipoprotein lipase, which represents the gatekeeper of triglyceride-rich lipoprotein metabolism. This dissertation describes a novel APOA5 mutation found in an 11-month old infant. The mutation led to an eight amino acid deletion within the coding region of the signal peptide. The loss of the eight amino acids resulted in dysfunction of the signal peptide, mainly because of the interrupted row of hydrophobic amino acids, which are usually building its hydrophobic core. The hydrophobic core of a signal peptide maintains the function to gate the nascent protein to the endoplasmatic reticulum to undergo the ca-nonical secretory pathway. To analyse the biochemical impairment of the mutation, we cloned a wild-type APOA5 construct into an expression vector, as well as rebuilt the mutation of the infant. The mutation was leading to an accumulation of APOA5 at the lipid droplets of hepatocytes, whereas the wild-type protein could be found at the plas-ma membrane of transfected primary hepatocytes. Subsequently, no secreted APOA5 could be detected within the supernatant of primary hepatocytes after transfection of the APOA5 construct coding the mutant gene. The deletion resulted in Hypertriglyceridemia, which was associated with acute ab-dominal pain. For the purpose of Hypertriglyceridemia, a homozygote mutation was needed, as the father and mother, both first degree cousins and heterozygote for the mutation, did not present severe Hypertriglyceridemia. The father had mildly increased triglyceride levels whereas the levels of the mother were within the reference range. Interestingly, both grandfathers – which also displayed a milder Hypertriglyceridemia - suffered in early heart attacks at the age of 45 and 51, leading to the presumption of an atherogenic influence of the mutation. The therapy options were at first to lower the amount of food intake derived lipids, which were taken up, so that the suggestion of weaning was enough to normalise the blood triglyceride level. The patient will have to fulfil a lifelong low-fat diet to decrease the risk of atherosclerosis and heart attack.