Reply
Authors: Silvia Sookoian, Carlos J. Pirola
Journal: Hepatology (2014)

Potential conflict of interest: Nothing to report. Author names in bold designate shared co‐first authorship. We thank Daly et al. for their interest in our study on the role of transmembrane 6 superfamily member 2 (TM6SF2) rs58542926 in nonalcoholic fatty liver disease (NAFLD). In our study, we confirmed and extended the original findings of Kozlitina et al., who reported a significant association of this variant with liver fat content.1 We further suggested that rs58542926 regulates liver transcript and protein expression in an allele‐specific manner.2 As Daly et al. noticed, neither we2 nor others3 observed an association with liver fibrosis; however, the variant was significantly associated with the degree of histological steatosis and nonalcoholic steatohepatitis (NASH).2,4 In fact, a large study from Italy showed that the association with fibrosis was completely abolished after adjusting for NASH.4 Daly et al. misunderstood the concept of "population stratification" in the 1000 Genomes; as shown in Fig. 1, there is a clear stratification in the European Ancestry sample that Liu et al. used as a control group.5 Population stratification and its effect in spurious allelic associations is largely known6; it does affect the frequency of allele variants, but it is not dependent on it.7 Daly et al. also raised some concerns about the controls we used: subjects whose ultrasonography and laboratory data indicated absence of disease. Conversely, the 1000 genome project used by Liu et al.5 contains no phenotypic data and the participants cannot be matched for sex or age.Figure 1: Linkage disequilibrium (LD) plot of TM6SF2 tagging the rs58542926. LD values were calculated by a pair‐wise estimation between SNPs genotyped in the same individuals and within a 100‐kb window. Figure illustrates differences among CEU, FIN, and GRB populations, confirming the assumption that given that the individuals collected belong to different subpopulations, spurious associations may be found between phenotypic traits and markers. Total European Ancestry (EUR) n = 379 (phase I sample) used by Liu et al. as controls5 is composed by: British from England and Scotland (GBR) n = 89; Finnish from Finland (FIN) n = 93; Iberian populations from Spain (IBS) n = 14; Toscani from Italy (TSI) n = 98; and Utah residents with Northern and Western European ancestry (CEU) n = 85. The samples for the 1000 Genomes Project are mostly anonymous and have no associated medical or phenotypic data (http://www.1000genomes.org/about), so inferences of subjects having or not fatty liver cannot be done in a reliable fashion. Available data suggest that the prevalence rate of NAFLD in the general European population is 2%‐44%,9 which means that in the 1000 genomes there are NAFLD subjects that would jeopardize the strength of the association reported by Liu et al.5In addition, we disagree that we have limited power to detect association with fibrosis. In fact, assuming the additive model of inheritance for an odds ratio of 2.9 (the one reported by Liu et at.5), and a realistic prevalence of advanced fibrosis of 20%, the power of our sample would be ∼99% for a P value = 0.05. In this association substudy, we did not use healthy controls as Daly et al mistakenly stated, but NAFLD patients proven by liver biopsy. Furthermore, we agree that rs58542926 is not a rare variant; however, it has been shown that homozygous TT subjects are, in fact uncommon, around the world.2 Finally, in our view, the association of the variant with fibrosis is still inconclusive. The assumption of Liu et al. attributing the variant a "fibrogenic" effect per se5 is difficult to reconcile with the knowledge gained on hepatic fibrogenesis as a wound‐healing response to repeated injury.8 Further experimental work is required to prove that rs58542926 is involved in fibrosis. Specifically, it would be very interesting to understand how the variant could possibly induce liver fibrosis without affecting the main disease phenotype, NASH/NAFLD as Liu t al. postulated.5