Increased Allele Frequency of GNPAT p.D519G in Compound HFE p.C282Y/p.H63D Heterozygotes with Elevated Serum Ferritin Levels

In hemochromatosis, iron overload is due to increased intestinal iron absorption attributable to mutations in several genes involved in the regulation of iron absorption and metabolism. The most common type of hemochromatosis is caused by mutations in the HFE gene, and homozygosity for the HFE p.C282Y mutation is associated with a risk of iron overload. Approximately 1:200 people of Caucasian origin are homozygous for p.C282Y, but only a minority of p.C282Y homozygotes develop significant iron overload. This is attributed in part to the presence of putative genetic modifiers of iron absorption. Recently, we identified GNPAT, encoding glyceronephosphate O-acyltransferase, as a potential modifier of HFE hemochromatosis in a whole-exome sequencing study of p.C282Y homozygotes with extreme iron overload phenotypes. A GNPAT polymorphism (p.D519G, rs11558492) was associated with severe iron overload among p.C282Y homozygous men (Hepatology 2015;62:429-39). Other studies have either substantiated or contradicted the importance of GNPAT p.D519G as a genetic modifier of iron phenotypes in HFE p.C282Y homozygotes. p.D519G is also a risk factor for familial (but not sporadic) porphyria cutanea tarda (Plos One 2016;11:e0163322). Some patients with hemochromatosis phenotypes are heterozygous for p.C282Y and a different HFE polymorphism, p.H63D (which is also common but usually not itself associated with clinically significant iron overload). Many p.C282Y/p.H63D compound heterozygotes have milder iron overload phenotypes than p.C282Y homozygotes. Herein, we report studies of the prevalence of p.D519G in a cohort of compound heterozygous p.C282Y/p.H63D Australian patients, with or without elevated serum ferritin (SF) levels. We also compared p.D519G allele frequency of the present p.C282Y/p.H63D compound heterozygotes with those of large population cohorts.

We identified 72 HFE p.C282Y/p.H63D compound heterozygotes with DNA available for analysis. Of these, 9 had missing SF data and were excluded. GNPAT p.D519G was assessed in the remaining 63 subjects. Compound heterozygotes with elevated SF levels ≥300 µg/mL (males) and ≥200 µg/mL (females) were selected as cases (n=28), and participants with SF levels below these cut-offs were classified as controls (n=35). The mean age at the time of testing was 31 y (males 31, females 31) in the control group and 48 y (males 47, females 51) in subjects with elevated SF levels. Among cases, mean SF was 612 µg/L (median 557 µg/L, IQR 361- 821 µg/L). Mean SF in the control group was 85 µg/L (median 58 µg/L, IQR 38 - 121 µg/L). All samples were genotyped for GNPAT p.D519G. We compared the p.D519G allele frequency of the present subjects, with and without elevated SF, to the p.D519G frequency in publically available datasets. p.D519G allele frequency was greater in the elevated SF group (37.5%) than the control group (24.3%), but this difference was not significant (p=0.1285). p.D519G was more prevalent in our cohort of p.C282Y/p.H63D compound heterozygotes with elevated SF (37.5%) than in European populations reported in public datasets: ExAC 19.7%, 1000G 21.3%, gnomAD 20.4%, and ESP 20.6%. There was a significant association between allele count and case/control status among men (type 3 analysis of effects; p = 0.049) but not women. For a male case, the odds of having either 1 or 2 alleles versus having 0 alleles was 5.08 (95% CI, 1.01, 25.66) times higher than that of a male control.

In conclusion, our results demonstrate that GNPAT p.D519G is associated with elevated SF levels in Australian HFE p.C282Y/p.H63D compound heterozygotes and that the p.D519G allele frequency is greater in p.C282Y/p.H63D compound heterozygotes with elevated SF than in several large European population cohorts. We found a statistically significant association between the allele count and the case/control status among men. The small number of subjects with elevated SF in our study may have limited our ability to demonstrate significant differences in some comparisons. Some subjects with p.D519G do not have elevated SF, suggesting that there are other factors, genetic or environmental, which also affect iron absorption in p.C282Y/p.H63D compound heterozygotes.