Exome Sequencing Identifies a GNPAT Variant Associated with Severe Iron Overload in HFE C282Y Homozygous Men with Extreme Phenotypes; Possible Role in Regulation of Hepcidin Expression

Variability in the severity of iron overload among homozygotes for the HFE C282Y polymorphism is one of the major unsolved problems in our understanding of hereditary hemochromatosis (HH). We previously conducted exome sequencing of DNA from 35 HFE C282Y male homozygotes with either markedly increased iron stores (n=22; cases) or normal to mildly increased iron stores (n=13; controls) to identify rare and common causal variants associated with variability of disease expression in HH. The 35 participants, residents of the U.S., Canada, and Australia, reported little or no alcohol consumption. Criteria for HFE C282Y homozygotes with increased iron stores included serum ferritin >1000 µg/L at diagnosis and either (a) hepatic iron concentration >236 µmol/g dry weight (reference range 0-36 µmol/g) or (b) mobilized body iron >10 g by quantitative phlebotomy. Criteria for HFE C282Y homozygotes with normal or mildly elevated iron stores included (a) serum ferritin <300 µg/L or either (a) age ≥40 y with ≤2.5 g iron removed by phlebotomy to achieve serum ferritin <50 µg/L, or (b) age ≥50 y with ≤3.0 g iron removed by phlebotomy. After quality control filtering, sequencing data included 82,068 single nucleotide variants and 1,403 insertions/deletions (indels); 10,337 genes were tested for a difference between cases and controls. We identified the polymorphism GNPAT p.D519G (1556A>G; exon 11; chromosome 1q42; rs11558492) as the most significantly different variant between cases and controls (p=0.033 by the likelihood ratio test after correction for multiple comparisons). In a principal components analysis of ancestry, all 35 study participants were clustered closely together within a larger group of Europeans. Mean (SD) ages at presentation were 53 (11.5) y and 57 (10.0) y for cases and controls, respectively. Median serum ferritin was 2391 µg/L in cases and 302 μg/L in controls. The median transferrin saturation (96%) was greater in cases than controls (70%).

Sixteen of 22 case participants had polymorphism GNPAT p.D519G (rs11558492) (15 heterozygotes, 1 homozygote); no control participant had this polymorphism. The homozygous case presented at age 26 y with severe iron overload but no cirrhosis. One GNPAT p.D519G heterozygote presented at age 36 y with severe iron overload and cirrhosis. GNPAT p.D519G is common among people of European descent (allele frequency 20.6%) and might interact with aberrant HFE to increase the risk of hepatic iron overload. More recently, we compared the allele frequencies of GNPAT p.D519G in the present 22 cases and 13 controls with that of 4300 European Americans in the NHLBI Exome Sequencing Project Exome Variant Server. The allele frequency in cases was greater than that of European Americans (38.6% vs. 20.6%, respectively; p = 0.0076). The allele frequency in controls was significantly lower (0% vs. 20.6%, p = 0.0054). Next, to determine whether other known mutations influenced iron phenotypes, the exome data were used to screen for mutations in HAMP, HJV, TFR2, FPN1, and TMPRSS6. One case participant was heterozygous for HJV p.G320V; he was among the six case participants who did not have GNPAT p.D519G. No other known or probable mutation that would possibly explain differences in expression between cases and controls was found.

To examine functional consequences of GNPAT deficiency, we now have performed siRNA-based knockdown of GNPAT in the human liver cell line HepG2/C3A. GNPAT was efficiently knocked down by its siRNA by ~85% compared to control siRNA as assayed by qPCR. This knockdown resulted in a >17-fold decrease in HAMP mRNA expression. mRNA expression of two genes coordinately regulated with HAMP, ID1 (inhibitor of DNA binding protein 1) and SMAD7 (SMAD family member 7), was similarly decreased, as was expression of phospho-SMAD 1/5/8, suggesting that GNPAT knockdown affects the baseline activity of the bone morphogenetic protein 6 (BMP6)-SMAD pathway.

Our data indicate that GNPAT p.D519G is associated with a high-iron phenotype in male HFE C282Y homozygotes and may participate in hepcidin regulation, thereby modifying severity of iron overload. The results identify GNPAT as a candidate gene for expanded studies to examine its function in regulating iron absorption and metabolism and to identify newly-diagnosed C282Y homozygotes whose risk for development of severe iron overload is great.