Our current systematic review and meta-analysis of 13 case-control studies did not find a significant association between the presence of HFE mutations and NAFLD in Caucasians. Specifically, we showed that the presence of iron overloading genotypes (C282Y/C282Y, C282Y/H63D) did not increase the risk of having NAFLD compared to controls. When we imputed controls from NHANES, we found similar inferences except from the presence of homozygosity. This may be explained by the presence of oversampling of cases with C282Y homozygosity among the case-only studies rather than a real association. In non Caucasian populations, our results based on three case-control studies suggest an excess of H63D mutants in NAFLD cases compared to controls.
In contrast to Ellervik et al [17
], our analyses showed no association due, we believe, to three key factors. First, applying the criteria they described, two studies should not have been excluded [45
], and, among the studies included, Ellervik et al. did not abstract the studies with the greatest number of cases [37
] in the meta-analysis. Secondly, weights used for meta-analysis appear to be inconsistent from the confidence intervals estimated by Ellervik and colleagues (Ellervik et al., Webfigures 6 and 9
). In other words, using the abstracted study-specific odds ratios and 99% confidence intervals (Webfigures 6 and 9
) we found incorrect weights and, consequently, inaccurate pooled odds ratios for the C282Y/C282Y genotypes and mixed heterozygotes, but not for the H63D/H63D genotype. Finally, two of the studies abstracted by Ellervik et al. did not genotype H63D in all individuals and this fact was not taken into account in the prior meta-analysis [20
]. An individual heterozygous for one mutation may also be heterozygous for another mutation (compound heterozygotes); therefore, it is possible that, if the reference group with genotype WT/WT may contain three other types of people, namely those with H63D/H63D, C282/H63D and H63D/WT. As a consequence, Ellervik et al. used incorrect study-specific odds ratios because they did not take into account this difference in the genotyping frequencies, and therefore, overestimated the pooled estimates for the C282Y/C282Y (OREllervik
:10.42, 99% CI: 2.05, 52.99; ORcorrected
: 5.87, 99% CI: 1.47, 23.48) and for the mixed heterozygotes genotypes (OREllervik
: 3.30, 99% CI: 1.40,7.70; ORcorrected
: 1.47, 99% CI: 1.01, 2.12). In contrast we found similar estimates for the H63D homozygotes (OREllervik
: 1.30, 99% CI: 0.49, 3.43; ORcorrected
:1.25, 99% CI: 0.50, 3.10) (Figure S5
). When we applied those corrections and replicated the Ellervik et al. meta-analysis focusing on their search period, we did not find any association between HFE
and NAFLD (data not shown).
The lack of power, different ethnic variation, information and selection biases and the presence of confounders have been frequently quoted to explain the inconsistency across HFE
-related studies [20
]. A meta-analysis is a possible solution to overcoming the issue of power. In our study of 1,727 NAFLD Caucasian cases and 4,275 controls, we had 88% power to detect an OR of 1.20 assuming a prevalence of any HFE
mutation in the reference population of 36.9% (estimated from our findings) and a two-sided alpha error of 0.05 [65
]. Therefore, it is likely that even if HFE
variants had an effect on the presence of NAFLD, the effect size would be relatively small. We also showed that information/selection biases did not substantially influence our results. Our results showed a trend to increased odds in non Caucasian populations, however, this finding deserves more studies since the overall estimate is driven by largest of the three studies[36
The current analysis has several limitations. First of all, we weren’t able to address whether the HFE
genetic variants acted as a disease-modifying gene in NASH (e.g. compare genotypes in simple steatosis only to those in NASH) or the impact of HFE
genetic variants on iron biomarkers in NAFLD patients. The published data were insufficient to address such question; and remains unsolved [58
]. Our definition of ethnicity was somehow arbitrary but, we believe, was consistent with the reported literature. It also reflects the unusual reporting of ethnicity across studies, a key variable in genetic studies. Third, the extrapolation of these results in the general population is somehow difficult because the majority of the case-control studies came from the hospital setting (where cases were defined using histology). Finally, when we examined statistical heterogeneity across different clinically relevant characteristics as previously described, our inference of no association between the presence of any HFE mutation and NAFLD remained unchanged. We acknowledged that we did not have enough characteristics of each study to correct for the presence of statistical heterogeneity. Only cooperation between researchers and individual data meta-analysis could address this question using meta-regression or genome wide association studies.
In contrast, our meta-analysis has several strengths. Not only we systemically reviewed the literature but also we obtained primary data from authors if needed. Compared to Ellervik, our current initiative focus and expands our knowledge on the association between the presence of HFE genetic variants and NAFLD.
In conclusion, we have found no association between the HFE genotype and NAFLD in Caucasians and non-Caucasians. We believe the HFE may have none or, at maximum, a marginal role in the development of NAFLD. Future studies should focus on the role of HFE in the progression of NAFLD and its association with iron biomarkers (peripheral and hepatic).