GNPAT polymorphism rs11558492 is not associated with increased severity in a large cohort of HFE p.Cys282Tyr homozygous patients
Authors: Dimitri Tchernitchko, Virginie Scotet, Thibaud Lefebvre, Carine L'Hostis, Isabelle Gourlaouen, Marie‐Christine Merour, Khadidja Rebah, Katell Peoc'h, Suzanne Assari, Claude Ferec, Hervé Puy, Gérald Le Gac
Journal: Hepatology (2016)

This work has been funded by the French Blood Agency (grants APR‐2010 and APR‐2013. Potential conflict of interest: Nothing to report. Author names in bold designate shared co‐first authorship. TO THE EDITOR: McLaren et al. recently used extreme phenotype sampling in a whole‐exome sequencing study to show that coinheritance of a common GNPAT polymorphism (rs11558492) in HFE p.Cys282Tyr homozygous males was associated with high‐iron phenotypes. Functional impact of glyceronephosphate O‐acyltransferase (GNPAT) deficiency in HepG2/C3A cells showed that small‐interfering‐RNA‐based knockdown of GNPAT significantly decreased hepcidin expression. The investigators argued that genotyping the nonsynonymous single‐nucleotide polymorphism, rs11558492, underlying p.Asp519Gly substitution, could help identify HFE‐related hemochromatosis patients at elevated long‐term risk of severe iron overload.1 To elucidate the prognostic value of GNPAT rs11558492, we addressed two important questions discussed by McLaren et al.: Impact of GNPAT rs11558492 in HFE p.Cys282Tyr homozygotes with intermediate iron phenotype: We recruited a large cohort of unrelated patients (n = 512) included in a phlebotomy program in a medical center of French Blood Agency. Main characteristics are presented in Table 1. Median serum ferritin (SF) was 633 μg/L (863 in males, 382 in females), and median amount of iron removed (AIR) by phlebotomy was 3.2 g (4.5 in males, 1.9 in females). Only 32 males (11.6%) and 5 females (2.1%) had >10 g of mobilized body iron. Linear regression revealed no significant association with log‐transformed SF and log‐transformed AIR, in contrast with positive effects of male sex, age at diagnosis, alcohol consumption, and body mass index (BMI) on both iron parameters. Results were unaffected by excluding females to eliminate sex bias in iron accumulation (Table 2). Impact of GNPAT rs11558492 on hepcidin synthesis: We selected 71 HFE p.Cys282Tyr homozygotes (35 males and 36 females) with serum sampled before phlebotomy. We eliminated patients with past or current alcohol consumption ≥20 g/day, attributed to the effect of alcohol on hepcidin expression in the liver. Serum hepcidin on liquid chromatography with tandem mass spectrometry did not differ significantly between patients harboring at least one variant allele versus wild‐type GNPAT (Supporting Fig. S1; nonparametric Mann‐Whitney U test). Table 1 - Sociodemographic and Biochemical Characteristics of the 512 p.Cys282Tyr Patients Whole (n = 512) Males (n = 276) Females (n = 236) Patient characteristics No. Frequency No. Frequency No. Frequency Sex Males 276 53.9% Females 236 46.1% Age at diagnosis (years) ≥60 109 21.3% 49 17.8% 60 25.4% [50‐60] 123 24.0% 59 21.4% 64 27.1% [40‐50] 129 25.2% 77 27.9% 52 22.0% <40 151 29.5% 91 33.0% 60 25.4% Means (± SD) 47.3 (13.6) 46.4 (12.7) 48.3 (14.5) Alcohol intake (g/day) ≥20 105 20.5% 85 30.8% 20 8.5% <20 407 79.5% 191 69.2% 216 91.5% Body mass index (kg/m2) ≥30 47 9.2% 26 9.4% 21 8.9% [25‐30] 148 28.9% 92 33.3% 56 23.7% <25 317 61.9% 158 57.2% 159 67.4% Means (± SD) 24.4 (4.1) 24.9 (3.9) 23.7 (4.2) Iron parameters Median IQR Median IQR Median IQR Transferrin saturation (%) 81 [68‐89] 84 [72‐92] 77 [60‐86] Serum ferritin (μg/L) 633 [366‐1,067] 863 [597‐1,612] 382 [227‐633] Amount of iron removed (g) 3.2 [1.8‐5.5] 4.5 [3.2‐7.0] 1.9 [1.1‐3.2] GNPAT rs11558492 No. Frequency No. Frequency No. Frequency Genotype Homozygous (T/T) 33 6.4% 17 6.2% 16 6.8% Heterozygous (G/T) 179 35.0% 94 34.1% 85 36.0% Wt/wt (G/G) 300 58.6% 165 59.8% 135 57.2% At least one variant allele 212 41.4% 111 40.2% 101 42.8% Wt/wt 300 58.6% 165 59.8% 135 57.2% Abbreviation: IQR, interquartile range. Table 2 - Regression Coefficients (β) and 95% Confidence Intervals (CIs) for Variables Influencing Serum Ferritin (μg/L; natural log transformation) and Iron Removed by Phlebotomy (g; natural log transformation) in p.Cys282Tyr Homozygous Patients Whole (n = 512) Males (n = 276) Serum ferritin Amount of iron removed Serum ferritin Amount of iron removed eβ 95% CI p eβ 95% CI p eβ 95% CI p eβ 95% CI p Sex Males 1.00 1.00 Females 0.39 [0.34‐0.45] <0.0001 0.40 [0.36 ‐1.20] <0.0001 Age at diagnosis (years) ≥60 1.59 [1.27‐1.99] <0.0001 1.43 [1.17‐1.75] 0.0006 1.50 [1.17‐1.91] 0.0013 1.36 [1.11‐1.67] 0.0036 [50‐60] 1.37 [1.10‐1.70] 0.0048 1.40 [1.15‐1.70] 0.0009 1.61 [1.28‐2.04] <0.0001 1.55 [1.27‐1.88] <0.0001 [40‐50] 1.25 [1.01‐1.55] 0.0442 1.35 [1.11‐1.64] 0.0026 1.22 [0.98‐1.51] 0.0716 1.27 [1.06‐1.52] 0.0101 <40 1.00 1.00 1.00 1.00 Alcohol intake (g/day) ≥20 2.23 [1.85‐2.68] <0.0001 2.23 [1.89‐2.63] <0.0001 1.65 [1.39‐1.97] <0.0001 1.68 [1.45‐1.94] <0.0001 <20 1.00 1.00 1.00 1.00 Body mass index (kg/m2) ≥30 1.25 [0.94‐1.65] 0.1260 1.11 [0.86‐1.43] 0.4291 0.96 [0.71‐1.29] 0.7648 0.94 [0.73‐1.22] 0.6410 [25‐30] 1.36 [1.13‐1.62] 0.0009 1.27 [1.08‐1.49] 0.0042 1.09 [0.90‐1.31] 0.3841 1.05 [0.90‐1.23] 0.5284 <25 1.00 1.00 1.00 1.00 GNPAT rs11558492 Homozygous (T/T) 1.10 [0.78‐1.53] 0.5928 1.15 [0.85‐1.56] 0.3557 2.50 [0.88‐1.27] 0.6209 1.28 [0.94‐1.73] 0.1190 Heteroygous (G/T) 0.97 [0.82‐1.15] 0.7298 1.00 [0.86‐1.17] 0.9856 1.06 [0.76‐1.58] 0.5580 1.11 [0.95‐1.30] 0.1713 wt/wt (G/G) 1.00 1.00 1.00 1.00 At least one variant allele 0.99 [0.84‐1.16] 0.8911 1.02 [0.88‐1.18] 0.7810 1.06 [0.89‐1.27] 0.4962 1.14 [0.98‐1.32] 0.0864 wt/wt 1.00 1.00 1.00 1.00 Exp(β) values correspond to multiplicative difference in the outcome variable for a level of the explanatory variable compared to the reference level. For example, exp(β) = 2.23 means that serum ferritin will be 2.23 times higher in patients consuming at least 20g alcohol per day than in patients with a lower alcohol intake. [Correction added November 22, 2016: Table note was added after original online publication.] Notably, the GNPAT rs11558492 variant allele showed exactly the same frequency (20.3%, very close to the 19.7% reported by the Exome Aggregation Consortium in 66,736 alleles from non‐Finnish Europeans) in 37 high expressers (male; daily alcohol consumption <20 g; normal BMI; SF >1,000 μg/L; AIR >5 g) and 38 low expressers (male; daily alcohol consumption <20 g; normal BMI; SF <700 μg/L; AIR <3 g). Three follow‐up studies published so far reported conflicting results, some associating GNPAT rs11558492 genotypes and exacerbated iron phenotypes, and others finding no GNPAT variant enrichment in HFE p.Cys282Tyr homozygous patients with well‐defined extreme phenotypes. The present study is the largest in unselected HFE patients, and suggests that a direct link between the GNPAT rs11558492 polymorphism and the phenotype expression of HFE p.Cys282Tyr homozygous patients, including serum hepcidin levels, is unlikely.