EDITORIAL Glyceronephosphate O-Acyltransferase as a Hemochromatosis Modifier Gene: Another Iron in the Fire? See Article on Page 429
H
ereditary hemochromatosis (HH) is a common genetic disorder characterized primarily by iron overload among Caucasians of Northern European origin.1-3 The most common form of the disease is hemochromatosis type 1, which is caused by mutations in the HFE gene, with the homozygous C282Y variant being the most common.2 Hemochromatosis type 2, 3, and 4 are less common and are caused by impaired functioning of various signaling molecules that are involved in the regulation of iron homeostasis, such as hemojuvelin (HJV) or hepcidin (HAMP) in the case of HH type 2, transferrin receptor (TfR) 2 (TfR2) in HH type 3, and ferroportin (FPN) in HH type 4, respectively.2 Hemochromatosis types 1-3 are associated with impaired production of hepcidin, the major iron-regulatory hormone.2 Hepcidin expression is regulated at the transcriptional level in the liver by body iron stores, based on a complex signaling mechanism that has still not been fully elucidated.1-3 Circulating hepcidin binds to FPN, which results in intracellular transport and lysosomal degradation of this protein.1 FPN is expressed on the basolateral aspect of intestinal absorptive cells; therefore, reduced levels of hepcidin result in increased iron absorption through iron transport across the intestinal epithelium.2 Population-based screening studies have demonstrated that only a minority of C282Y homozygotes fully express the hemochromatosis phenotype, suggest-
Abbreviations: BMP, bone morphogenic protein; FPN, ferroportin; GNPAT, glyceronephosphate O-acyltransferase; HH, hereditary hemochromatosis; HJV, hemojuvelin; siRNA, small interfering RNA; SMAD, small mothers against decapentaplegic; SNVs, single-nucleotide variations; TfR, transferrin receptor. Received March 19, 2015; accepted March 26, 2015. Address reprint requests to: Kris V. Kowdley, M.D., Liver Care Network and Organ Care Research, 1101 Madison Street, Suite 200, Seattle, WA 98104. E-mail: [email protected]; fax: 11-206-320-7431. 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.27813 Potential conflict of interest: Nothing to report.
ing that there are genetic and environmental factors that can modify the severity and penetrance of the disease phenotype.4,5 In its most severe form, HFEassociated hemochromatosis is characterized by greatly increased body iron stores, cirrhosis, hepatocellular carcinoma, cardiomyopathy, endocrinopathy, and/or arthritis.3,6,7 Among patients expressing the iron overload phenotype, early initiation of therapeutic phlebotomy treatment can prevent organ damage associated with progressive iron accumulation.3,6,7 Therefore, a better understanding of the various molecular players involved in the regulation of hepcidin and iron homeostasis is crucial to identifying genetic modifiers that could influence the degree of iron overload and disease severity in hereditary hemochromatosis. In the current issue of HEPATOLOGY, McLaren et al. performed exome sequencing in HFE C282Y homozygous subjects in order to identify variants that might modify the severity of the iron overload phenotype.8 Male homozygous HFE C282Y subjects displaying extremes of the iron overload phenotype, as determined by serum ferritin concentration, liver iron concentration, and the amount of iron removed by therapeutic phlebotomy to achieve iron depletion, were included in this study. Using the above criteria, the researchers selected high iron phenotypes (n 5 22; cases) and low iron phenotypes (n 5 13; controls). After screening over 82,068 singlenucleotide variants (SNVs) and a total of 10,337 genes for differences between cases and controls, they found that a novel variant in the glyceronephosphate O-acyltransferase (GNPAT) gene showed the most significant association with severe iron overload. Though none of the low-iron-phenotype controls showed this polymorphism, 15 of the 22 high-iron-phenotype subjects displayed a heterozygous polymorphism in GNPAT at position D519G (GNPATp.D519G). GNPAT8,9 is a peroxisomal enzyme responsible for the first step in the biosynthesis of lipid molecules called plasmalogens, one of the main types of ether phospholipids in humans.9 These molecules are an essential component of cell membranes.9 Although 337
338
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Fig. 1. Proposed role for GNPAT in iron sensing and hepcidin signling. The figure shows our current knowledge of the complex mechanism whereby circulating iron as well as membrane-bound HFE and other membrane-bound and cytoplasmic non-HFE-signaling proteins (TfRs, HJV, BMP-SMAD-signaling proteins, Il-6/IL6R, among others) regulate hepcidin transcription. As shown in the figure, GNPAT, a peroxisomal enzyme, may play a role in regulation of hepcidin through transferrin receptor cycling between the peroxisome and plasma membrane surface, or may directly affect hepcidin expression, possibly through an unknown cytoplasmic effector or through an unidentified pathway. Abbreviation: IL, interleukin.
their exact role is unclear, they are thought to be involved in essential functions, such as maintaining membrane fluidity, taking part in signal transduction processes, providing defense against oxidative stress, and membrane fusion.9 A deficiency in GNPAT is associated with impaired membrane trafficking with altered cholesterol distribution and formation of clathrin-coated pits, resulting in impaired TfR recycling.9 It has also been demonstrated that the D519G mutation in GNPAT causes a 70% reduction in its enzymatic activity.9 Rare, naturally occurring dysfunctional mutations in the GNPAT gene (but not GNPAT.p.D519G) have been previously described in humans, resulting in a genetic disorder called rhizomelic chondrodysplasia punctate type 2, which is associated with skeletal dysplasia and severe mental retardation.10 However, iron overload has not been shown to be associated with these rare diseases.8 Other GNPAT variants have been found in subjects with peroxisomal disorders, a class of disease in which hepatic iron overload is observed.8,9
To further elucidate the mechanism by which GNPAT mutation may result in iron overload, McLaren et al. utilized a human hepatoma cell line, HepG2/C3A, to knock down GNPAT expression and examine its effect on hepcidin signaling. GNPAT knockdown led to a >17-fold down-regulation of hepcidin expression. In order to shed light on the signaling pathway involved in GNPAT-mediated regulation of hepcidin, the researchers next examined whether bone morphogenic protein/small mothers against decapentaplegic (BMP-SMAD) signaling was required for GNPAT to exert its effect on hepcidin expression. They assessed phosphorylation of SMAD1/5/ 8, a downstream effector of BMP signaling1-3 in GNPAT-deficient (cells treated with small interfering RNA [siRNA] to GNPAT) and GNPAT-proficient cells (cells treated with control siRNA), with or without exogenous BMP6 addition. They found that in the absence of BMP6, levels of phosphorylated SMAD1/5/8 were low, whereas there was no effect of GNPAT silencing on phospho-SMAD1/5/8 levels in the presence of BMP6. These findings suggest that GNPAT deficiency has no
HEPATOLOGY, Vol. 62, No. 2, 2015
effect on the BMP-SMAD pathway, clarifying that GNPAT exerts its effect on hepcidin independent of BMP-SMAD signaling. However, the researchers did not determine whether GNPAT deficiency has any effect on TfR recycling and whether this led to reduced hepcidin expression levels, leaving a question to be answered by future mechanistic studies. Although this study demonstrates that the coinheritance of GNPAT.p.D519G in some males with HFE C282Y homozygosity is associated with a more severe iron phenotype and highlights a potential prognostic role for GNPAT.p.D519G polymorphism in predicting iron-related disease, there are several pieces of the puzzle that remain to be addressed. The researchers were able to show a decrease in hepcidin expression upon GNPAT silencing in hepatocytes, suggesting the involvement of GNPAT in transcriptional regulation of hepcidin, but the mechanism of that downregulation is not known. Does GNPAT regulate hepcidin transcription through its traditional enzymatic role of peroxisomal membrane biosynthesis and TfR turnover, or does it utilize a novel mechanism involving an unidentified cytoplasmic factor, or is it through an unknown pathway (Fig. 1)? The findings from this study suggest that the loss of GNPAT expression or SNVs in the GNPAT expression/ activity affect hepcidin expression and that GNPAT is a genetic modifier of the hemochromatosis iron phenotype. Although hepcidin expression is inappropriately low in HFE C282Y homozygotes, leading to increased body iron stores, increased ferritin and iron overload in hemochromatosis patients with diseasemodifying genes suggest that further down-regulation is possible.8,11 Thus, GNPAT polymorphisms, such as GNPAT.pD519G, could cause a further reduction in hepcidin levels, thereby increasing iron overload. Identifying GNPAT.pD519G variants in young C282Y hemochromatosis patients might be predictive of potential overload in later life and prove useful to recommend effective clinical management of the disease. The demonstration of GNPATp.D519G as a significant phenotype modifier of HFE C282Y homozygosity provides new data that modifier alleles could be a prominent source of variable disease penetrance. Finally, given that 6 of the 22 high-ironphenotype subjects in this study did not display the
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coinheritance of GNPAT.pD519G, these results point to the possible discovery of other novel phenotypemodifying variants that could affect clinical expression of HFE hemochromatosis. Acknowledgment: The authors thank Dr. Sunil Thomas from the University of Washington at Seattle for assistance with the figure and careful reading of the manuscript. PRIYA HANDA, PH.D. KRIS V. KOWDLEY, M.D.
Liver Care Network and Organ Care Research Swedish Medical Center Seattle, WA
References 1. Ganz T. Systemic iron homeostasis. Physiol Rev 2013;93:1721-1741. 2. De Domenico I, McVey Ward D, Kaplan J. Regulation of iron acquisition and storage: consequences for iron-linked disorders. Nat Rev Mol Cell Biol 2008;9:72-781. 3. Weiss G. Genetic mechanisms and modifying factors in hereditary hemochromatosis. Nat Rev Gastroenterol Hepatol 2010;7:50-58. 4. Beutler E, Felitti VJ, Koziol JA, Ho NJ, Gelbart T. Penetrance of 845G–>A (C282Y) HFE hereditary haemochromatosis mutation in the USA. Lancet. 2002 Jan 19;359(9302):211-8. 5. Allen KJ, Gurrin LC, Constantine CC, Osborne NJ, Delatycki MB, Nicoll AJ, et al. Iron-overload-related disease in HFE hereditary hemochromatosis. N Engl J Med 2008;358:221-230. 6. Kanwar P, Kowdley KV. Diagnosis and treatment of hereditary hemochromatosis: an update. Expert Rev Gastroenterol Hepatol 2013;7:517530. Erratum in: Expert Rev Gastroenterol Hepatol 2013;7:767. 7. Bacon BR, Adams PC, Kowdley KV, Powell LW, Tavill AS; American Association for the Study of Liver Diseases. Diagnosis and management of hemochromatosis: 2011 practice guideline by the American Association for the Study of Liver Diseases. HEPATOLOGY 2011;54:328-343. 8. 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. 9. Thai TP, Rodemer C, Jauch A, Hunziker A, Moser A, Gorgas K, Just WW. Impaired membrane traffic in defective ether lipid biosynthesis. Hum Mol Genet 2001;10:127-136. 10. Itzkovitz B, Jiralerspong S, Nimmo G, Loscalzo M, Horovitz DD, Snowden A, et al. Functional characterization of novel mutations in GNPAT and AGPS, causing rhizomelic chondrodysplasia punctata (RCDP) types 2 and 3. Hum Mutat 2012;33:189-197. 11. Benyamin B, Esko T, Ried JS, Radhakrishnan A, Vermeulen SH, Traglia M, et al. Novel loci affecting iron homeostasis and their effects in individuals at risk for hemochromatosis. Nat Commun 2014;5:4926.
Author names in bold designate shared co-first authorship.
H
ereditary hemochromatosis (HH) is a common genetic disorder characterized primarily by iron overload among Caucasians of Northern European origin.1-3 The most common form of the disease is hemochromatosis type 1, which is caused by mutations in the HFE gene, with the homozygous C282Y variant being the most common.2 Hemochromatosis type 2, 3, and 4 are less common and are caused by impaired functioning of various signaling molecules that are involved in the regulation of iron homeostasis, such as hemojuvelin (HJV) or hepcidin (HAMP) in the case of HH type 2, transferrin receptor (TfR) 2 (TfR2) in HH type 3, and ferroportin (FPN) in HH type 4, respectively.2 Hemochromatosis types 1-3 are associated with impaired production of hepcidin, the major iron-regulatory hormone.2 Hepcidin expression is regulated at the transcriptional level in the liver by body iron stores, based on a complex signaling mechanism that has still not been fully elucidated.1-3 Circulating hepcidin binds to FPN, which results in intracellular transport and lysosomal degradation of this protein.1 FPN is expressed on the basolateral aspect of intestinal absorptive cells; therefore, reduced levels of hepcidin result in increased iron absorption through iron transport across the intestinal epithelium.2 Population-based screening studies have demonstrated that only a minority of C282Y homozygotes fully express the hemochromatosis phenotype, suggest-
Abbreviations: BMP, bone morphogenic protein; FPN, ferroportin; GNPAT, glyceronephosphate O-acyltransferase; HH, hereditary hemochromatosis; HJV, hemojuvelin; siRNA, small interfering RNA; SMAD, small mothers against decapentaplegic; SNVs, single-nucleotide variations; TfR, transferrin receptor. Received March 19, 2015; accepted March 26, 2015. Address reprint requests to: Kris V. Kowdley, M.D., Liver Care Network and Organ Care Research, 1101 Madison Street, Suite 200, Seattle, WA 98104. E-mail: [email protected]; fax: 11-206-320-7431. 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.27813 Potential conflict of interest: Nothing to report.
ing that there are genetic and environmental factors that can modify the severity and penetrance of the disease phenotype.4,5 In its most severe form, HFEassociated hemochromatosis is characterized by greatly increased body iron stores, cirrhosis, hepatocellular carcinoma, cardiomyopathy, endocrinopathy, and/or arthritis.3,6,7 Among patients expressing the iron overload phenotype, early initiation of therapeutic phlebotomy treatment can prevent organ damage associated with progressive iron accumulation.3,6,7 Therefore, a better understanding of the various molecular players involved in the regulation of hepcidin and iron homeostasis is crucial to identifying genetic modifiers that could influence the degree of iron overload and disease severity in hereditary hemochromatosis. In the current issue of HEPATOLOGY, McLaren et al. performed exome sequencing in HFE C282Y homozygous subjects in order to identify variants that might modify the severity of the iron overload phenotype.8 Male homozygous HFE C282Y subjects displaying extremes of the iron overload phenotype, as determined by serum ferritin concentration, liver iron concentration, and the amount of iron removed by therapeutic phlebotomy to achieve iron depletion, were included in this study. Using the above criteria, the researchers selected high iron phenotypes (n 5 22; cases) and low iron phenotypes (n 5 13; controls). After screening over 82,068 singlenucleotide variants (SNVs) and a total of 10,337 genes for differences between cases and controls, they found that a novel variant in the glyceronephosphate O-acyltransferase (GNPAT) gene showed the most significant association with severe iron overload. Though none of the low-iron-phenotype controls showed this polymorphism, 15 of the 22 high-iron-phenotype subjects displayed a heterozygous polymorphism in GNPAT at position D519G (GNPATp.D519G). GNPAT8,9 is a peroxisomal enzyme responsible for the first step in the biosynthesis of lipid molecules called plasmalogens, one of the main types of ether phospholipids in humans.9 These molecules are an essential component of cell membranes.9 Although 337
338
HANDA AND KOWDLEY
HEPATOLOGY, August 2015
Fig. 1. Proposed role for GNPAT in iron sensing and hepcidin signling. The figure shows our current knowledge of the complex mechanism whereby circulating iron as well as membrane-bound HFE and other membrane-bound and cytoplasmic non-HFE-signaling proteins (TfRs, HJV, BMP-SMAD-signaling proteins, Il-6/IL6R, among others) regulate hepcidin transcription. As shown in the figure, GNPAT, a peroxisomal enzyme, may play a role in regulation of hepcidin through transferrin receptor cycling between the peroxisome and plasma membrane surface, or may directly affect hepcidin expression, possibly through an unknown cytoplasmic effector or through an unidentified pathway. Abbreviation: IL, interleukin.
their exact role is unclear, they are thought to be involved in essential functions, such as maintaining membrane fluidity, taking part in signal transduction processes, providing defense against oxidative stress, and membrane fusion.9 A deficiency in GNPAT is associated with impaired membrane trafficking with altered cholesterol distribution and formation of clathrin-coated pits, resulting in impaired TfR recycling.9 It has also been demonstrated that the D519G mutation in GNPAT causes a 70% reduction in its enzymatic activity.9 Rare, naturally occurring dysfunctional mutations in the GNPAT gene (but not GNPAT.p.D519G) have been previously described in humans, resulting in a genetic disorder called rhizomelic chondrodysplasia punctate type 2, which is associated with skeletal dysplasia and severe mental retardation.10 However, iron overload has not been shown to be associated with these rare diseases.8 Other GNPAT variants have been found in subjects with peroxisomal disorders, a class of disease in which hepatic iron overload is observed.8,9
To further elucidate the mechanism by which GNPAT mutation may result in iron overload, McLaren et al. utilized a human hepatoma cell line, HepG2/C3A, to knock down GNPAT expression and examine its effect on hepcidin signaling. GNPAT knockdown led to a >17-fold down-regulation of hepcidin expression. In order to shed light on the signaling pathway involved in GNPAT-mediated regulation of hepcidin, the researchers next examined whether bone morphogenic protein/small mothers against decapentaplegic (BMP-SMAD) signaling was required for GNPAT to exert its effect on hepcidin expression. They assessed phosphorylation of SMAD1/5/ 8, a downstream effector of BMP signaling1-3 in GNPAT-deficient (cells treated with small interfering RNA [siRNA] to GNPAT) and GNPAT-proficient cells (cells treated with control siRNA), with or without exogenous BMP6 addition. They found that in the absence of BMP6, levels of phosphorylated SMAD1/5/8 were low, whereas there was no effect of GNPAT silencing on phospho-SMAD1/5/8 levels in the presence of BMP6. These findings suggest that GNPAT deficiency has no
HEPATOLOGY, Vol. 62, No. 2, 2015
effect on the BMP-SMAD pathway, clarifying that GNPAT exerts its effect on hepcidin independent of BMP-SMAD signaling. However, the researchers did not determine whether GNPAT deficiency has any effect on TfR recycling and whether this led to reduced hepcidin expression levels, leaving a question to be answered by future mechanistic studies. Although this study demonstrates that the coinheritance of GNPAT.p.D519G in some males with HFE C282Y homozygosity is associated with a more severe iron phenotype and highlights a potential prognostic role for GNPAT.p.D519G polymorphism in predicting iron-related disease, there are several pieces of the puzzle that remain to be addressed. The researchers were able to show a decrease in hepcidin expression upon GNPAT silencing in hepatocytes, suggesting the involvement of GNPAT in transcriptional regulation of hepcidin, but the mechanism of that downregulation is not known. Does GNPAT regulate hepcidin transcription through its traditional enzymatic role of peroxisomal membrane biosynthesis and TfR turnover, or does it utilize a novel mechanism involving an unidentified cytoplasmic factor, or is it through an unknown pathway (Fig. 1)? The findings from this study suggest that the loss of GNPAT expression or SNVs in the GNPAT expression/ activity affect hepcidin expression and that GNPAT is a genetic modifier of the hemochromatosis iron phenotype. Although hepcidin expression is inappropriately low in HFE C282Y homozygotes, leading to increased body iron stores, increased ferritin and iron overload in hemochromatosis patients with diseasemodifying genes suggest that further down-regulation is possible.8,11 Thus, GNPAT polymorphisms, such as GNPAT.pD519G, could cause a further reduction in hepcidin levels, thereby increasing iron overload. Identifying GNPAT.pD519G variants in young C282Y hemochromatosis patients might be predictive of potential overload in later life and prove useful to recommend effective clinical management of the disease. The demonstration of GNPATp.D519G as a significant phenotype modifier of HFE C282Y homozygosity provides new data that modifier alleles could be a prominent source of variable disease penetrance. Finally, given that 6 of the 22 high-ironphenotype subjects in this study did not display the
HANDA AND KOWDLEY
339
coinheritance of GNPAT.pD519G, these results point to the possible discovery of other novel phenotypemodifying variants that could affect clinical expression of HFE hemochromatosis. Acknowledgment: The authors thank Dr. Sunil Thomas from the University of Washington at Seattle for assistance with the figure and careful reading of the manuscript. PRIYA HANDA, PH.D. KRIS V. KOWDLEY, M.D.
Liver Care Network and Organ Care Research Swedish Medical Center Seattle, WA
References 1. Ganz T. Systemic iron homeostasis. Physiol Rev 2013;93:1721-1741. 2. De Domenico I, McVey Ward D, Kaplan J. Regulation of iron acquisition and storage: consequences for iron-linked disorders. Nat Rev Mol Cell Biol 2008;9:72-781. 3. Weiss G. Genetic mechanisms and modifying factors in hereditary hemochromatosis. Nat Rev Gastroenterol Hepatol 2010;7:50-58. 4. Beutler E, Felitti VJ, Koziol JA, Ho NJ, Gelbart T. Penetrance of 845G–>A (C282Y) HFE hereditary haemochromatosis mutation in the USA. Lancet. 2002 Jan 19;359(9302):211-8. 5. Allen KJ, Gurrin LC, Constantine CC, Osborne NJ, Delatycki MB, Nicoll AJ, et al. Iron-overload-related disease in HFE hereditary hemochromatosis. N Engl J Med 2008;358:221-230. 6. Kanwar P, Kowdley KV. Diagnosis and treatment of hereditary hemochromatosis: an update. Expert Rev Gastroenterol Hepatol 2013;7:517530. Erratum in: Expert Rev Gastroenterol Hepatol 2013;7:767. 7. Bacon BR, Adams PC, Kowdley KV, Powell LW, Tavill AS; American Association for the Study of Liver Diseases. Diagnosis and management of hemochromatosis: 2011 practice guideline by the American Association for the Study of Liver Diseases. HEPATOLOGY 2011;54:328-343. 8. 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. 9. Thai TP, Rodemer C, Jauch A, Hunziker A, Moser A, Gorgas K, Just WW. Impaired membrane traffic in defective ether lipid biosynthesis. Hum Mol Genet 2001;10:127-136. 10. Itzkovitz B, Jiralerspong S, Nimmo G, Loscalzo M, Horovitz DD, Snowden A, et al. Functional characterization of novel mutations in GNPAT and AGPS, causing rhizomelic chondrodysplasia punctata (RCDP) types 2 and 3. Hum Mutat 2012;33:189-197. 11. Benyamin B, Esko T, Ried JS, Radhakrishnan A, Vermeulen SH, Traglia M, et al. Novel loci affecting iron homeostasis and their effects in individuals at risk for hemochromatosis. Nat Commun 2014;5:4926.
Author names in bold designate shared co-first authorship.
