Studying an Lp(a) mutation in human RNA and liver organoids

Background: About one fifth of the population has high plasma concentrations of the so-called lipoprotein(a) [Lp(a)] particle which is associated with an increased cardiovascular risk. Nonetheless, many aspects of the Lp(a) metabolism are still poorly understood. We recently identified a very frequent mutation in the LPA gene (named 4925G>A), which reduces Lp(a) by 70% and potentially explains many of the until now unclear aspects of this trait. However, its precise effects could not be analyzed yet, because no optimal cell culture or animal model exists so far. All available cell culture systems present important shortcomings, which severely hamper Lp(a) research. Lp(a) is expressed exclusively in the liver and is present only in humans and apes. More than 40 different variants of Lp(a) exist in t population (isoforms). So called organoids represent a new model in cell biology, which recapitulate the features of true organs much better than normal cell culture models and thus represent a promising system to study Lp(a). However, the Lp(a) isoform and production of existing organoid models are unknown. Aims: This project pursues two complementary aims: investigating the effects of 4925G>A and creating a bank of liver organoids, which produce several different Lp(a) isoforms and which can so be used as a new and state-of-the-art resource for Lp(a) research. Methods: We will collect a small amount of healthy liver tissue from voluntary donors undergoing liver resection. About 20% are expected to carry the 4925G>A mutation. We will use gene sequencing methods, which analyze all gene products in the cell simultaneously (transcriptom sequencing), to study the effects of 4925G>A on the Lp(a) production in these samples. Moreover, we will study whether so called transcript-negative null alleles, i.e. gene variants, which do not produce any Lp(a) at all and which have been observed in baboons, exist also in humans and investigate their genetic causes. Finally, we will create a comprehensive bank of at least 50 liver organoids with defined Lp(a) isoforms and determine the culture parameters necessary to achieve Lp(a) expression from this liver organoids. Opportunities: Understanding the effects of KIV-2 4925G>A effects on mRNA can point towards novel Lp(a)-lowering mechanisms, as also the identification of human transcript-null alleles and underlying genetic variants will do. Conversely, a collection of Lp(a) producing liver organoids with defined Lp(a) characteristics will represent, for the first time, a research model, which is very close to true human livers. They provide new opportunities for Lp(a) research and testing of Lp(a) treatment. No comparable model exists to date.

The lipoprotein(a) concentration in the blood is one of the most important risk factors for cardiovascular disease in the population. Lp(a) concentrations vary in the population by about 1000-foldand, unlike other blood lipids such as LDL or HDL cholesterol, are almost completely genetically regulated. This occurs through a complex interaction of a so-called copy number variation, the KIV-2 repeat, and many different genetic mutations ("genetic variants"). A copy number variation is a region in the genome that occurs at a different number in each person. The number of KIV-2 elements determines about 30-70% of the variance of Lp(a) in the blood. However, there are many common genetic variants in the population that influence the Lp(a) value additionally. In this project, we described the two most important genetic Lp(a) variants in the European population and, in particular, characterized the "KIV-2 4925G>A variant" in more detail. We showed that the KIV-2 4925G>A variant causes or strongly modifies the effect of two other genetic variants. In earlier studies, an Lp(a)-reducing effect of the so-called "pentanucleotide variant" was found, but its mechanism was never elucidated. We were able to show that the pentanucleotide variant only reflects the effect of 4925G>A and has no own effect. Furthermore, we could show that the effect of the Thr3888Pro variant, another frequently studied Lp(a) variant, depends on the presence of KIV-2 4925G>A. When KIV-2 4925G>A is present, Thr3888Pro is associated with a lower Lp(a) value. However, when Thr3888Pro is present alone, it actually increases the Lp(a) level. This was previously unknown and sheds new light on the possible molecular effects of Thr3888Pro. It also shows how complex the interaction of the different genetic variants in Lp(a) is and that the KIV-2 variant 4925G>A plays a particularly important role in the genetic regulation of Lp(a) levels in blood. A further aim of the project was to cultivate a new liver cell model in the form of organoids in order to investigate Lp(a) metabolism. In the course of the experiments, we found that it is very important that the organoid cells are not only grown as liver cells, but also have the correct "zonal identity". The human liver is divided into so-called "lobules", which in turn are subdivided into different zones. Each zone has different functions, activates different genes and produces different substances. In which of these zones Lp(a) is formed was previously unknown. Therefore an important piece of the puzzle for establishing new cell culture models to study Lp(a) metabolism was missing. In extensive experiments with several different methods, we were able to define the hepatic zone in which Lp(a) is probably produced. This provides an important new piece of the puzzle for understanding Lp(a) metabolism.