CORRESPONDENCE GNPAT Variant Associated With Severe Iron Overload in HFE Hemochromatosis To the Editor: By using exome sequencing in highly selected patients and in vitro experiments, the elegant study by McLaren et al. demonstrated the rs11558492 polymorphism in the GNPAT gene as a major factor of phenotypic expression in HFE hemochromatosis.1 As stated by the investigators, it is necessary to confirm a prognostic role for GNPAT p.D519G in a large sample of C282Y homozygotes. In the recent genome-wide association study that we conducted in unselected C282Y homozygotes with HFE hemochromatosis, the rs11558492 was part of the polymorphisms studied and, surprisingly, was not found as a significant genetic modifier.2 We went back to our data and specifically studied markers of iron burden according to the genotype distribution of rs11558492 in the whole population, including females (n 5 474), and in two subgroups consisting (1) of all males (n 5 274) and (2) males selected using strictly similar criteria to those from McLaren et al. (no past or current alcohol consumption 20g/day; n 5 36). Ancestry was genetically examined and all patients were identified as Europeans and particularly as coming from Western Europe.2 Allele frequencies of GNPAT p.D519G were homogeneous across both the whole population and subcohorts 1 and 2 (26.6%, 26.3%, and 25% respectively; Fisher’s test, P 5 0.974). They were similar with those from the 35 patients of McLaren et al. (24.3%; P 5 0.78) and from the British in England and Scotland population of the 1000Genome (24.7%; P 5 1.00). However, they were greater than that of European Americans from the Exome Variant Server database (20.6%; P < 0.001). Using regression models adjusted for age and sex for the whole population and age for males subgroups 1 and 2, we assessed the association between GNPAT rs11558492 genotypes, iron burden, and severe fibrosis defined as Metavir stages 3 and

4. Linear regression failed to show any significant association with serum ferritin (P 5 0.847, 0.232, and 0.166, respectively) or amount of iron removed (P 5 0.827, 0.21, and 0.688, respectively). Logistic regression did not find any significant association with severe fibrosis in any of these groups (P 5 0.579, 0.15, and 0.943, respectively). Iron burden, according to serum ferritin or amount of iron removed, was similar in hetero- or homozygous patients for rs11558492, irrespectively of the population studied (Fig. 1). Finally, we cannot extend, in our population, the results of McLaren et al. because our data show that GNPAT p.D519G is not associated with iron burden or fibrosis in an unselected French population of C282Y homozygotes. More studies with diverse populations are needed to shed light on these discrepancies. EDOUARD BARDOU-JACQUET, M.D., PH.D.1,2,3* MARIE DE TAYRAC, PH.D.2,4,5* JEAN MOSSER, PH.D.2,4,5 YVES DEUGNIER, M.D.1,2,3 1 CHU Rennes French Reference Center for Rare Iron Overload Diseases of Genetic Origin Rennes, France 2 Universit e Rennes 1 Rennes, France 3 INSERM UMR 991 Rennes, France 4 CNRS UMR 6290 Rennes, France

Fig. 1. Mean ferritin at diagnosis (A) and amount of iron removed (B) levels in the different GNPAT rs11558492 genotype categories. A/G and G/G genotypic categories are pooled and standard deviations are reported as vertical bars. 1917

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5 CHU Rennes Service de Ge ne tique Mol e culaire et Genomique Rennes, France

References 1. McLaren CE, Emond MJ, Subramaniam VN, Phatak PD, Barton JC, Adams PC, et al. Exome sequencing in HFE C282Y homozygous men with extreme phenotypes identifies a GNPAT variant associated with severe iron overload. HEPATOLOGY 2015;62:429-439. 2. de Tayrac M, Roth M, Jouanolle A, Coppin H, le Gac G, Piperno A, et al. Genome-wide association study identifies TF as a significant modifier gene of iron metabolism in HFE hemochromatosis. J Hepatol 2015; 62:664-672.

HEPATOLOGY, December 2015

Six homozygotes with severe iron overload in our study did not have p.D519G, suggesting that other modifier alleles remain undiscovered. It is unknown whether GNPAT p.D519G protein, or a closely linked locus, augments iron absorption. However, a larger, multinational study that we are presently conducting should be informative. Any results may also differ from those in France owing to haplotypic and broader genetic differences between populations. We agree with Bardou-Jacquet et al. that: “More studies with diverse populations are needed to shed light on these discrepancies.”1 CHRISTINE E. MCLAREN, PH.D.1* MARY J. EMOND, PH.D.2* V. NATHAN SUBRAMANIAM, PH.D.3,4* PRADYUMNA D. PHATAK, M.D.5 JAMES C. BARTON, M.D.6 PAUL C. ADAMS, M.D.7 LAWRIE W. POWELL, M.D., PH.D.3,4,8 LYLE C. GURRIN, PH.D.9 GRANT A. RAMM, PH.D.3,4 GREGORY J. ANDERSON, PH.D.3,10 GORDON D. MCLAREN, M.D.11,12 1 Department of Epidemiology University of California Irvine, CA 2 Department of Biostatistics University of Washington Seattle, WA 3 QIMR Berghofer Medical Research Institute Brisbane, Australia 4 Faculty of Medicine and Biomedical Sciences The University of Queensland Brisbane, Australia 5 Rochester General Hospital Rochester, NY 6 Southern Iron Disorders Center Birmingham, AL 7 Department of Medicine London Health Sciences Center London, Ontario, Canada 8 Royal Brisbane & Women’s Hospital Brisbane, Australia 9 Center for MEGA Epidemiology The University of Melbourne Melbourne, Australia 10 School of Medicine and School of Chemistry and Molecular Bioscience University of Queensland St. Lucia, Australia 11 Department of Veterans Affairs Long Beach Healthcare System Long Beach, CA 12 Division of Hematology/Oncology Department of Medicine University of California Irvine, CA

Author names in bold designate shared co-first authorship. C 2015 by the American Association for the Study of Liver Diseases. Copyright V View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.27854 *These authors contributed equally to this work. Potential conflict of interest: Nothing to report.

Reply: We appreciate the response of Bardou-Jacquet et al. to our report of a positive association of GNPAT p.D519G (rs11558492) with severe iron overload in males with HFE C282Y homozygosity and low alcohol consumption.1 Their genome-wide association study of 474 unselected French male and female C282Y homozygotes found no significant association of p.D519G with iron overload. Briefly, these differences in our studies can be linked to the design and power of our study and to inclusion criteria.

Power Considerations The power of our extreme phenotypes study should be greater than that of many larger clinical studies.2 Bardou-Jacquet et al. do not provide power calculations nor confidence intervals (CIs) with effect sizes for association with serum ferritin (SF). Biologically/ clinically significant effect sizes could be covered by 95% CIs. As with others,3 we observed a strong positive association between SF and alcohol consumption. Adjusting for alcohol provides greater power to detect associations with modifier alleles, but it is not apparent whether Bardou-Jacquet et al. made such an adjustment. Their subset of 36 males who consumed