The patatin-like phospholipase domain containing 3 (PNPLA3) gene (viz. its I148M variant) is one of the key players in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). We have identified a novel insertion/deletion variant of 1114 bp, localized in the second intron of the PNPLA3 gene, which corresponds to the 3' terminal sequence of the long-interspersed element (LINE-1). DNA analysis of 122 NAFLD patients and 167 control subjects as well as RNA analysis of 19 liver biopsies revealed that the novel variant is very common (frequency = 0.41), fully linked to the clinically important I148M variant, and clinically silent. Although the LINE-1 insertion does not seem to have any biological effect, it can impede genotyping of the I148M variant. If insertion prevents the attachment of the diagnostic primer, then the non-insertion allele will be selectively amplified; and thus the frequency of the 148M "risk" allele will be significantly overestimated due to the complete linkage of the LINE-1 insertion and the 148I allele of the PNPLA3 gene. Therefore, our findings underline the importance of careful design and consistent documentation of the methodology, including primer sequences. Critical revisions of the results of some studies that have already been reported may therefore be needed.

4th Department of Internal Medicine 1st Faculty of Medicine and General University Hospital Prague Charles University U Nemocnice 499 2 12808 Prague Czech Republic

Department of Gastroenterology and Hepatology Regional Hospital Liberec Husova 357 10 46063 Liberec Czech Republic

Department of Microbiology and Biological Defence Research Military Health Institute Prague Tychonova 1 16001 Prague Czech Republic

Institute of Medical Biochemistry and Laboratory Diagnostics 1st Faculty of Medicine and General University Hospital Prague Charles University Kateřinská 32 12108 Prague Czech Republic

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Younossi Z, et al. Global perspectives on nonalcoholic fatty liver disease and nonalcoholic steatohepatitis. Hepatology. 2019;69:2672–2682. doi: 10.1002/hep.30251. PubMed DOI

Romeo S, et al. Genetic variation in PNPLA3 confers susceptibility to nonalcoholic fatty liver disease. Nat. Genet. 2008;40:1461–1465. doi: 10.1038/ng.257. PubMed DOI PMC

Buch S, et al. A genome-wide association study confirms PNPLA3 and identifies TM6SF2 and MBOAT7 as risk loci for alcohol-related cirrhosis. Nat. Genet. 2015;47:1443–1448. doi: 10.1038/ng.3417. PubMed DOI

He S, et al. A sequence variation (I148M) in PNPLA3 associated with nonalcoholic fatty liver disease disrupts triglyceride hydrolysis. J. Biol. Chem. 2010;285:6706–6715. doi: 10.1074/jbc.M109.064501. PubMed DOI PMC

Chen W, Chang B, Li L, Chan L. Patatin-like phospholipase domain-containing 3/adiponutrin deficiency in mice is not associated with fatty liver disease. Hepatology. 2010;52:1134–1142. doi: 10.1002/hep.23812. PubMed DOI PMC

Basantani MK, et al. Pnpla3/Adiponutrin deficiency in mice does not contribute to fatty liver disease or metabolic syndrome. J. Lipid Res. 2011;52:318–329. doi: 10.1194/jlr.M011205. PubMed DOI PMC

Smagris E, et al. Pnpla3I148M knockin mice accumulate PNPLA3 on lipid droplets and develop hepatic steatosis. Hepatology. 2015;61:108–118. doi: 10.1002/hep.27242. PubMed DOI PMC

BasuRay S, Smagris E, Cohen JC, Hobbs HH. The PNPLA3 variant associated with fatty liver disease (I148M) accumulates on lipid droplets by evading ubiquitylation. Hepatology. 2017;66:1111–1124. doi: 10.1002/hep.29273. PubMed DOI PMC

Yang A, Mottillo EP, Mladenovic-Lucas L, Zhou L, Granneman JG. Dynamic interactions of ABHD5 with PNPLA3 regulate triacylglycerol metabolism in brown adipocytes. Nat. Metab. 2019;1:560–569. doi: 10.1038/s42255-019-0066-3. PubMed DOI PMC

BasuRay S, Wang Y, Smagris E, Cohen JC, Hobbs HH. Accumulation of PNPLA3 on lipid droplets is the basis of associated hepatic steatosis. Proc. Natl. Acad. Sci. 2019;116:9521–9526. doi: 10.1073/pnas.1901974116. PubMed DOI PMC

Tilson SG, et al. Modelling PNPLA3-associated non-alcoholic fatty liver disease using human induced pluripotent stem cells. Hepatology. 2021 doi: 10.1002/hep.32063. PubMed DOI PMC

Beck CR, Garcia-Perez JL, Badge RM, Moran JV. LINE-1 elements in structural variation and disease. Annu. Rev. Genom. Hum. Genet. 2011;12:187–215. doi: 10.1146/annurev-genom-082509-141802. PubMed DOI PMC

Scott AF, et al. Origin of the human L1 elements: Proposed progenitor genes deduced from a consensus DNA sequence. Genomics. 1987;1:113–125. doi: 10.1016/0888-7543(87)90003-6. PubMed DOI PMC

Grimaldi G, Skowronski J, Singer MF. Defining the beginning and end of KpnI family segments. EMBO J. 1984;3:1753–1759. doi: 10.1002/j.1460-2075.1984.tb02042.x. PubMed DOI PMC

Han JS, Szak ST, Boeke JD. Transcriptional disruption by the L1 retrotransposon and implications for mammalian transcriptomes. Nature. 2004;429:268–274. doi: 10.1038/nature02536. PubMed DOI

Chalasani N, et al. The diagnosis and management of nonalcoholic fatty liver disease: Practice guidance from the American Association for the Study of Liver Diseases. Hepatology. 2018;67:328–357. doi: 10.1002/hep.29367. PubMed DOI

Dvorak K, et al. Use of non-invasive parameters of non-alcoholic steatohepatitis and liver fibrosis in daily practice—An exploratory case-control study. PLoS One. 2014;9:e111551. doi: 10.1371/journal.pone.0111551. PubMed DOI PMC

Subhanova I, et al. Expression of Biliverdin Reductase A in peripheral blood leukocytes is associated with treatment response in HCV-infected patients. PLoS One. 2013;8:e57555. doi: 10.1371/journal.pone.0057555. PubMed DOI PMC

Urbanek P, et al. No association of promoter variations of HMOX1 and UGT1A1 genes with liver injury in chronic hepatitis C. Ann. Hepatol. 2011;10:445–451. doi: 10.1016/S1665-2681(19)31511-X. PubMed DOI

den Dunnen JT, et al. HGVS recommendations for the description of sequence variants: 2016 Update. Hum. Mutat. 2016;37:564–569. doi: 10.1002/humu.22981. PubMed DOI

Koren MJ, Hunninghake DB. Clinical outcomes in managed-care patients with coronary heart disease treated aggressively in lipid-lowering disease management clinics The alliance study. J. Am. Coll. Cardiol. 2004;44:1772–1779. PubMed

Shi YY, He L. SHEsis, a powerful software platform for analyses of linkage disequilibrium, haplotype construction, and genetic association at polymorphism loci. Cell Res. 2005;15:97–98. doi: 10.1038/sj.cr.7290272. PubMed DOI

Li, Z. et al. A partition-ligation-combination-subdivision EM algorithm for haplotype inference with multiallelic markers: update of the SHEsis (http://analysis.bio-x.cn). Cell. Res.19, 519–523. 10.1038/cr.2009.33 (2009). PubMed