**4. Genetic Tests**

Genome wide association studies increased our knowledge and understanding of genetic and genomic alterations and components during NASH development and progression leading to the identification of several potential target genes, not only for therapeutic intervention, but also for the prediction of risk patients. The most abundant alterations are genetic variations in form of single nucleotide polymorphisms (SNPs). As very common alterations in NASH variants of the genes encoding patatin-like phospholipase domain-containing protein 2 (*PNPLA3*), transmembrane 6 superfamily member 2 (*TM6SF2*), membrane-bound O-acetyltransferase domain-containing protein 7 (*MBOAT7*) and glucokinase regulatory protein (*GCKR*) were identified. These genes are spread across the human genome (Figure 4).

**Figure 4.** Gene alterations in NASH. Gene alterations/modifications associated with the pathogenesis of NASH affect the *GCKR*, *TM6SF2*, *PNPLA3*, and *MBOAT7* genes. The chromosomal location of respective genes is depicted in the ideogram. Gene annotations were done with the Genome Decoration Page (https://www.ncbi.nlm.nih.gov/genome/tools/gdp) and genomic coordinates deposited in the Online Mendelian Inheritance in Man (OMIM) database under accession no. 609567 (*PNPLA3*, 22q13.31), 606563 (*TM6SF2*, 19p13.11), 606048 (*MBOAT7*, 19q13.42), and 600842 (*GCKR*, 2p23.3).

The patatine-like phospholipase domain-containing protein 3 gene (*PNPLA3*) encodes for the triacylglycerol lipase adiponutrin that mediates the hydrolysis of triacylglycerol in adipocytes and hepatocytes. One of the most abundant DNA sequence variations associated with NAFLD and NASH is the isoleucine to methionine substitution in the *PNPLA3* gene at position 148 (PNPLA3-148M variant) [128,129]. This leads to a loss-of-function ending up in the accumulation of mutated 148M in hepatocytes and hepatic stellate cells where it further leads to the malignant storage of triglycerides [130]. Respective patients that are homozygous and carrying this variant have a tenfold increased risk to develop NAFLD-related HCC.

Transmembrane 6 superfamily member 2 (*TM6SF2*) is generally expressed in the liver and in the small intestine. It regulates the secretion of triglycerides and the content of lipid droplets as it is involved in VLDL (very low-density lipoprotein) secretion. The most frequent SNP is known in the *TM6SF2* gene is the E167K variant. This polymorphism is a loss-of-function mutation triggered by the replacement of glutamic acid by lysine at position 167. This mutation leads to the accumulation of triglycerides in hepatocytes and at the same time lowers the systemic lipoprotein levels [131].

A mutation leading to the replacement of cysteine by threonine is a common variant within the membrane bound O-acyltransferase domain-containing 7 (*MBOAT7*) gene. Recent investigations link this variant with a decrease in systemic and intrahepatic phosphatidyl-inositol containing arachidonic acid, thereby leading to an increased risk of getting NAFLD, NASH, and related end-stage liver diseases [132]. Another genetic variant, which was shown to be directly associated with the development of NAFLD by influencing the regulation of *de novo* lipogenesis and hepatic glucose uptake is the P446L mutation in the *GCKR* gene encoding for the glucokinase regulatory protein [133].

Besides those mentioned above, further genome-wide analysis show other gene variants associated with a higher risk of developing NAFLD and progress to related end-stage diseases. In this context, genetic polymorphisms of ethanol metabolizing enzymes (e.g., alcohol dehydrogenase) and cytochrome p450 2E1 (*CYP2E1*) activation triggering oxidative biotransformation and ROS formation, which is relevant in generating lipid peroxides, and their interference with the outcome of alcohol-induced liver disease and NASH has been discussed [134]. In particular, there are several clinical studies showing

that alterations in *CYP2E1* activity are observed under various conditions, including obesity rendering respective persons more susceptible to liver injury [134,135]. Getting more knowledge about the exact effects influenced by those gene variations can be beneficial in the development of new therapeutic options and drug targets to treat NASH in the future.

More recently, several exploratory studies conducted in preclinical models identified circulating levels of non-coding RNA (ncRNA) such as microRNA and long ncRNA (lncRNA) to be associated with the pathogenesis and progression of various liver diseases [136]. In particular, several microRNAs representing a new class of highly conserved small non-coding RNA were shown to be critically involved in the regulation of complex gene networks in almost all acute and chronic liver disease [137]. As an example, profiling in diet-induced NASH progression and regression models identified the upregulation of a signature composed of six defined microRNA in NASH mice that allowed accurate distinguishing of NASH from lean mice [138]. However, in view of the large number of reported preclinical studies on miRNA, only a few have entered clinical trials and precise information about their diagnostic and prognostic value for human liver disease is still missing.
