The Immunogenetics of Hereditary Hemochromatosis CHRISTOPHER F. BRYAN, PHD
ABSTRACT: Hereditary hemochromatosis (HH), an iron overload disease caused by unregulated intestinal iron absorption, is a recessive HLAlinked disease. HH is the most common inherited metabolic disorder with one of every 400 to 500 individuals having both genes and being likely to develop the disease. Thus, although the product of the hemochromatosis gene is unknown, its mode of inheritance allows HLA-genotyping of the proband and his/her siblings to be highly predictive of the genetic propensity to develop the clinical features of HH. In view of the known immunoregulatory properties of iron and its binding proteins, it is important to determine if the high levels of storage iron in HH influence the immunosurveillance network in HH patients and whether that has any clinical relevance. We have defined certain alterations of the effector cells of the cellular arm of the immune system and have studied a patient with HH who had specific immune alterations, including delayed cutaneous-type hypersensitivity anergy, and was diagnosed with poorly differentiated adenocarcinoma of the stomach four years after his HH diagnosis. Those findings are consistent with the interpretation that in certain clinical situations of elevated body iron stores, the immunoregulatory balance or environment may be tipped in favor of growth and development of cancer cells. KEY INDEXING TERMS: Cancer; HLA; Hemochromatosis; Immunosurveillance; Iron. [Am J Med Sci 1991; 301(1):47-49.]
T
he first objective of this discussion is to review the mode of inheritance of the gene(s) responsible for hereditary hemochromatosis. Secondly, I will present evidence that specific immune alterations occur in patients with hereditary hemochromatosis and that The author thanks, Beth Dahlsten for her excellent assistance' in the preparation of this manuscript. From the Midwest Organ Bank, Westwood, Kansas. Reprint requests: Dr. Christopher F. Bryan, Midwest Organ Bank, 1900 West 47th Place, Suite 400, Westwood, KS 66205. THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
certain ones are associated with the elevated level of iron that occurs in such patients. In humans, two inherited diseases involve inappropriate iron absorption, atransferanemia, and hereditary hemochromatosis (HH). The subsequent discussion in this paper will be confined to HH since so few cases of atransferanemia have been reported. The gene(s) responsible for the development of HH is linked to the major histocompatibility complex in man, HLA. The HLA genetic region has been extensively evaluated regarding its role in regulation of immune response and cellular interaction. By current estimates, HH is thought to be one of the more commonly inherited diseases. Such estimates suggest that one of every 400-500 individuals possesses both hemochromatosis genes and that one in every 1015 persons is a carrier of the hemochromatosis gene.! Hereditary hemochromatosis is an iron overload disorder that results from inappropriate and unregulated intestinal iron absorption. The absorbed iron is transported through the intestinal cell to the iron transport molecule transferrin, which distributes iron to the various tissues of the body. A gradual accumulation of iron thus occurs in various organs, especially in the liver, heart, and pancreas. Such iron overload in tissue generally results in clinically detectable sequelae, which become noticeable in men during the fourth or fifth decade in life and later in women. The diagnosis of HH is made by demonstration of iron deposition primarily in the parenchymal cells of the liver after other secondary causes have been ruled out. Although Sheldon presented familial evidence supporting the genetic nature of HH in 1935, vigorous debate ensued during the following 40 years regarding whether the etiology of HH was environmental or genetic. In 1976, Simon demonstrated that the HLA Class I antigens, A3 and B7, were both significantly associated with HH.2 That finding has been confirmed by every investigative group that has evaluated HLA antigens in HH, including our study in Louisiana with 15 probands that had been diagnosed with hereditary hemochromatosis. We identified the HLA-A3 antigen in 73% of our patients andHLA-B7 in 44%, compared to 23% and 17% of our controls, respectively. The next important finding was the definition of the mode of inheritance for the hemochromatosis gene. Si47
Hereditary HemochromcJtosis
mon showed that the gene(s) responsible for the development of hemochromatosis was found to be recessive and linked to the HLA-A locus. 1 That gives us a genetic model whereby siblings of an hemochromatosis patient may be genotyped with respect to their hemochromatosis genetic makeup. For example, if a sibling and a hemochromatosis patient are HLA genotypically identical, the sibling would possess both of the HLA-linked hemochromatosis gene(s) and is likely to eventually develop the disease. Alternatively, siblings who are HLA haploidentical with the hemochromatosis patient should only possess one hemochromatosis gene and most likely would not develop any clinical sequela of HH. Finally, siblings who did not share any HLA haplotype with their sibling should not possess a hemochromatosis gene. I present one of our family HLA genotype studies (Table 1). The proband, WL, was diagnosed with hereditary hemochromatosis by a liver biopsy. HLA genotyping of WL and seven siblings showed that she and her brother, BS, were HLA genotypically identical. The brother (BS) had a ferritin of 790 nanograms/ml, a transferrin saturation of 44%, and a liver biopsy that revealed significant iron deposition in the parenchymal cells in the absence of cirrhosis. In contrast, two other siblings, AM and LB, who shared only one haplotype with the proband did not have biochemically detectable increased iron stores. In that recessive and HLA-linked model, it is assumed that a recombination, between the hemochromatosis gene and HLA did not occur. The Salt Lake City group has identified a family wherein a recombination did occur between the hemochromatosis gene and the HLA-A and HLA-B loci,3 which has enabled the tentative mapping of the hemochromatosis gene centromeric to the HLA-A locus. Finally, in spite of our knowledge of the recessive mode of inheritance of the hemochromatosis gene and its linkage to the HLA complex, we still are unaware of the basic function and structure of that gene with respect to iron absorption. However, several questions remain to be answered regarding expression of the hemochromatosis gene, such as: 1. What is the nature of the gene product(s) of the hemochromatosis gene? 2. How is that gene and/or gene product involved in iron absorption?
3. Why is the hemochromatosis gene, a gene presumed to regulate iron absorption, located within the HLA gene complex, which encodes molecules involved in antigen presentation? 4. Is the molecular structure of the hemochromatosis gene related to that of HLA Class I genes? 5. What is the tissue distribution of the hemochromatosis gene product? Our challenge in the future is to understand the functional and structural aspects of the hemochromatosis gene product(s). Immunoregulatory Properties in Hereditary Hemochromatosis
The essential role of the immune system is to safeguard and maintain the integrity of the organism. The most striking feature of the immune response, whether cellular or humoral, is its exquisite specificity. This specificity is, however, relative rather than absolute. A second property of the immune response is that of heterogeneity, which exists at a variety of cellular and molecular levels. The immunoregulatory properties of iron and its binding proteins have been investigated most definitively in vitro. These studies have shown that iron and certain iron-binding proteins influence structural and functional properties of T and B lymphocytes. Furthermore, a variety of immunological abnormalities have been reported in patients with iron deficiency and iron overload. The initial studies that defined the immunoregulatory nature of iron examined the influence of high levels of iron on expression of lymphocyte surface molecules. In the first study, the T cell E-rosette receptor (CD2) was shown to be modulated by iron. 4 A subsequent study extended that observation and showed that mitoticallyactive lymphocytes had iron-modified expression of the T cell surface markers CD3 and CD4, which were decreased in expression by iron; expression of the transferrin receptor was increased by iron, and expression ofCD8, OKT10, HLA Class II antigens, and the thermostable erythrocyte-rosette receptor were not influenced by iron.5 Support for the postUlate that iron exerts certain immunoregulatory properties at the cellular level grew. Studies were performed that evaluated the effect of iron on functional in vitro immunological responses. One
Table 1. HLA Typing of a Family With One Proband With Hemochromatosis Family Member Evaluated
Age
WL (Proband)! BS (Brother) AM (Brother) LB (Sister)
69 53 67 76
!
HLA Genotype A3 A3 A3 A2
B62 Cw3 / A2 B27 Cw2 B62 Cw3 / A2 B27 Cw2 B62 Cw3 / A2 B27 Cw4 B44 Cw- / A2 B62 Cw4
Shared HLA Haplotypes
Ferritin 2
Biopsy-proven Hemochromatosis
2 1 1
1761 790 54 69
Yes Yes No No
The proband (WL) was diagnosed and this study conducted in 1982, at LSU Medical Center. normal range is 10-287 nanograms/ml; values for proband WL and BS were obtained at diagnosis.
2 Ferritin
48
January 1991 Volume 301 Number 1
Bryan
example was the effect of iron on the mixed lymphocyte culture (MLC) response, which measures the proliferative capacity of CD4 helper T cells in response to disparate HLA Class II antigens. Iron inhibited the proliferative response differentially with respect to HLA Class I phenotype in that the MLC response of individuals who possessed HLA,-A2 was resistant to the inhibitory influence of iron, whereas, inhibition was evident in the MLC response of those who lacked HLA-A2.6 A vast body of literature has accumulated regarding the relationship of iron with susceptibility to infections, and inflammatory and neoplastic diseases. It seems likely that the ability of microorganisms to compete successfully with the host for iron is a feature ofpathogenicity or virulence, and the ability of the host to limit availability of iron to the pathogens is associated with resistance to infection. In view of the potential in vivo immunoregulatory effects of iron,4-s we have evaluated patients with hereditary hemochromatosis to determine if quantitative or functional components of their immune system differed with respect to iron status. 7•S At least two observations in one such studys were consistent with but do not prove the postulate, that the elevated level of storage iron has an in vivo immunoregulatory influence. First, the absolute number of CDS positive T cells were significantly elevated in untreated hereditary hemochromatosis patients and reduced in treated patients, as compared with controls. Second, the proliferative response of peripheral blood mononuclear cells from untreated hereditary hemochromatosis patients to mitogens and in the mixed lymphocyte culture was suboptimal, whereas the response of peripheral blood mononuclear cells from treated hereditary hemochromatosis patients was normal. Immunoglobulin secretion by peripheral blood mononuclear cells from treated hereditary hemochromatosis patients was altered as compared to controls.s Finally, increased expression of the T cell surface receptor that forms thermostable erythrocyte-rosettes (TE-R) was noted. The TE-R receptor is normally expressed on thymocytes or activated T cells and not mature T cells. There was no association of TE-R receptor expression with iron status. 7 Taken together, these data define specific immune alterations in patients with hereditary hemochromatosis with certainty, consistent with the interpretation that cellular immunity may be influenced by the high level of storage iron in patients with hereditary hemochromatosis. Summary
In view of the incidence of hereditary hemochromatosis being close to one individual per 500 and the observation that the incidence of liver cancer in patients with HH is 219 times that of control populations,9 the diagnosis of
THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
HH and its subsequent treatment in the pre-cirrhotic stage is imperative. Although the gene(s) responsible for development of HH is unknown, the hemochromatosis gene is known to be recessive and linked to the HLA gene complex. Therefore, HLA genotyping in families where HH has been diagnosed to determine the linear arrangement of the HLA antigens in the proband may be an appropriate diagnostic adjunct in an effort to identify siblings who are in the pre-cirrhotic stage of hereditary hemachromatosis. Although a milange of immunologic abnormalities have been documented in patients with iron deficiency and in iron overload states, the clinical importance of such abnormalities remains largely undefined. Despite the less clear, clinically important immunologic consequences of iron overload, the immunoregulatory role of iron and its binding proteins clearly has been established and in the future should provide further important insights into the function of the immune system. One of many possible mechanisms that may, in part, explain the high incidence of liver cancer in hereditary hemochromatosis may be related to an effect of iron on the immunosurveillance network, such that in certain clinical situations of high body-iron storage, the immunoregulatory balance may be modified to create an environment that is more conducive to the growth and development of cancer cells. We are in the process of evaluating that postulate. References 1. Edwards CQ, Skolnick MH, Kushner JP: Hereditary hemochromatosis contributions of genetic analyses. Prog Bemat 7: 43-71, 1981. 2. Simon M, Bourel M, Fauchet R, Genetet B: Association of HLAA3 and HLA-BI4 antigens with idiopathic hemochromatosis. Gut 17:332-334, 1976. 3. Edwards CQ, Griffen LM, Dadone MM, Skolnick MH, Kushner JP: Mapping the locus for hereditary hemochromatosis: Localization between HLA-B and HLA-A. Am J Bum Genet 38:805811,1986. 4. de Sousa M, NishiyaK: Inhibition of E-Rosette formation by two iron salts. Cell Immunol38:203-208, 1978. 5. Bryan CF, Leech SH, Bozelka B: The immunoregulatory nature of iron. II. Lymphocyte surface marker expression. J Leukoc Biol 40:589-600, 1986. 6. Bryan CF, Nishiya K, Pollack MS, Dupont B, de Sousa M: Differential inhibition of the MLR by iron: Association with HLA phenotype. Immunogenetics 12:129-140, 1981. 7. Bryan CF, Leech SH, Ducos R, Edwards CQ, KushnerJP, Skolnick MH, Bozelka B, Linn JC, Gaumer R: Thermostable erythrocyte rosette-identification in peripheral blood. J Clin Immunol 4:2,1984. 8. Bryan CF, Leech SH, Kumar P, Gaumer R, Bozelka B, Morgan J: The immune system in hereditary hemochromatosis: A quantitative and functional assessment of the cellular arm. Am J Med Sci 301(1):55-61, 1991. 9. Niederau C, Fischer R, Sonnenberg A, Stremmel W, Trampisch HJ, Strohmeyer G: Survival and causes of death in cirrhotic and in noncirrhotic patients with primary hemochromatosis. N Engl J Med 313:1256-1262, 1985.
49
ABSTRACT: Hereditary hemochromatosis (HH), an iron overload disease caused by unregulated intestinal iron absorption, is a recessive HLAlinked disease. HH is the most common inherited metabolic disorder with one of every 400 to 500 individuals having both genes and being likely to develop the disease. Thus, although the product of the hemochromatosis gene is unknown, its mode of inheritance allows HLA-genotyping of the proband and his/her siblings to be highly predictive of the genetic propensity to develop the clinical features of HH. In view of the known immunoregulatory properties of iron and its binding proteins, it is important to determine if the high levels of storage iron in HH influence the immunosurveillance network in HH patients and whether that has any clinical relevance. We have defined certain alterations of the effector cells of the cellular arm of the immune system and have studied a patient with HH who had specific immune alterations, including delayed cutaneous-type hypersensitivity anergy, and was diagnosed with poorly differentiated adenocarcinoma of the stomach four years after his HH diagnosis. Those findings are consistent with the interpretation that in certain clinical situations of elevated body iron stores, the immunoregulatory balance or environment may be tipped in favor of growth and development of cancer cells. KEY INDEXING TERMS: Cancer; HLA; Hemochromatosis; Immunosurveillance; Iron. [Am J Med Sci 1991; 301(1):47-49.]
T
he first objective of this discussion is to review the mode of inheritance of the gene(s) responsible for hereditary hemochromatosis. Secondly, I will present evidence that specific immune alterations occur in patients with hereditary hemochromatosis and that The author thanks, Beth Dahlsten for her excellent assistance' in the preparation of this manuscript. From the Midwest Organ Bank, Westwood, Kansas. Reprint requests: Dr. Christopher F. Bryan, Midwest Organ Bank, 1900 West 47th Place, Suite 400, Westwood, KS 66205. THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
certain ones are associated with the elevated level of iron that occurs in such patients. In humans, two inherited diseases involve inappropriate iron absorption, atransferanemia, and hereditary hemochromatosis (HH). The subsequent discussion in this paper will be confined to HH since so few cases of atransferanemia have been reported. The gene(s) responsible for the development of HH is linked to the major histocompatibility complex in man, HLA. The HLA genetic region has been extensively evaluated regarding its role in regulation of immune response and cellular interaction. By current estimates, HH is thought to be one of the more commonly inherited diseases. Such estimates suggest that one of every 400-500 individuals possesses both hemochromatosis genes and that one in every 1015 persons is a carrier of the hemochromatosis gene.! Hereditary hemochromatosis is an iron overload disorder that results from inappropriate and unregulated intestinal iron absorption. The absorbed iron is transported through the intestinal cell to the iron transport molecule transferrin, which distributes iron to the various tissues of the body. A gradual accumulation of iron thus occurs in various organs, especially in the liver, heart, and pancreas. Such iron overload in tissue generally results in clinically detectable sequelae, which become noticeable in men during the fourth or fifth decade in life and later in women. The diagnosis of HH is made by demonstration of iron deposition primarily in the parenchymal cells of the liver after other secondary causes have been ruled out. Although Sheldon presented familial evidence supporting the genetic nature of HH in 1935, vigorous debate ensued during the following 40 years regarding whether the etiology of HH was environmental or genetic. In 1976, Simon demonstrated that the HLA Class I antigens, A3 and B7, were both significantly associated with HH.2 That finding has been confirmed by every investigative group that has evaluated HLA antigens in HH, including our study in Louisiana with 15 probands that had been diagnosed with hereditary hemochromatosis. We identified the HLA-A3 antigen in 73% of our patients andHLA-B7 in 44%, compared to 23% and 17% of our controls, respectively. The next important finding was the definition of the mode of inheritance for the hemochromatosis gene. Si47
Hereditary HemochromcJtosis
mon showed that the gene(s) responsible for the development of hemochromatosis was found to be recessive and linked to the HLA-A locus. 1 That gives us a genetic model whereby siblings of an hemochromatosis patient may be genotyped with respect to their hemochromatosis genetic makeup. For example, if a sibling and a hemochromatosis patient are HLA genotypically identical, the sibling would possess both of the HLA-linked hemochromatosis gene(s) and is likely to eventually develop the disease. Alternatively, siblings who are HLA haploidentical with the hemochromatosis patient should only possess one hemochromatosis gene and most likely would not develop any clinical sequela of HH. Finally, siblings who did not share any HLA haplotype with their sibling should not possess a hemochromatosis gene. I present one of our family HLA genotype studies (Table 1). The proband, WL, was diagnosed with hereditary hemochromatosis by a liver biopsy. HLA genotyping of WL and seven siblings showed that she and her brother, BS, were HLA genotypically identical. The brother (BS) had a ferritin of 790 nanograms/ml, a transferrin saturation of 44%, and a liver biopsy that revealed significant iron deposition in the parenchymal cells in the absence of cirrhosis. In contrast, two other siblings, AM and LB, who shared only one haplotype with the proband did not have biochemically detectable increased iron stores. In that recessive and HLA-linked model, it is assumed that a recombination, between the hemochromatosis gene and HLA did not occur. The Salt Lake City group has identified a family wherein a recombination did occur between the hemochromatosis gene and the HLA-A and HLA-B loci,3 which has enabled the tentative mapping of the hemochromatosis gene centromeric to the HLA-A locus. Finally, in spite of our knowledge of the recessive mode of inheritance of the hemochromatosis gene and its linkage to the HLA complex, we still are unaware of the basic function and structure of that gene with respect to iron absorption. However, several questions remain to be answered regarding expression of the hemochromatosis gene, such as: 1. What is the nature of the gene product(s) of the hemochromatosis gene? 2. How is that gene and/or gene product involved in iron absorption?
3. Why is the hemochromatosis gene, a gene presumed to regulate iron absorption, located within the HLA gene complex, which encodes molecules involved in antigen presentation? 4. Is the molecular structure of the hemochromatosis gene related to that of HLA Class I genes? 5. What is the tissue distribution of the hemochromatosis gene product? Our challenge in the future is to understand the functional and structural aspects of the hemochromatosis gene product(s). Immunoregulatory Properties in Hereditary Hemochromatosis
The essential role of the immune system is to safeguard and maintain the integrity of the organism. The most striking feature of the immune response, whether cellular or humoral, is its exquisite specificity. This specificity is, however, relative rather than absolute. A second property of the immune response is that of heterogeneity, which exists at a variety of cellular and molecular levels. The immunoregulatory properties of iron and its binding proteins have been investigated most definitively in vitro. These studies have shown that iron and certain iron-binding proteins influence structural and functional properties of T and B lymphocytes. Furthermore, a variety of immunological abnormalities have been reported in patients with iron deficiency and iron overload. The initial studies that defined the immunoregulatory nature of iron examined the influence of high levels of iron on expression of lymphocyte surface molecules. In the first study, the T cell E-rosette receptor (CD2) was shown to be modulated by iron. 4 A subsequent study extended that observation and showed that mitoticallyactive lymphocytes had iron-modified expression of the T cell surface markers CD3 and CD4, which were decreased in expression by iron; expression of the transferrin receptor was increased by iron, and expression ofCD8, OKT10, HLA Class II antigens, and the thermostable erythrocyte-rosette receptor were not influenced by iron.5 Support for the postUlate that iron exerts certain immunoregulatory properties at the cellular level grew. Studies were performed that evaluated the effect of iron on functional in vitro immunological responses. One
Table 1. HLA Typing of a Family With One Proband With Hemochromatosis Family Member Evaluated
Age
WL (Proband)! BS (Brother) AM (Brother) LB (Sister)
69 53 67 76
!
HLA Genotype A3 A3 A3 A2
B62 Cw3 / A2 B27 Cw2 B62 Cw3 / A2 B27 Cw2 B62 Cw3 / A2 B27 Cw4 B44 Cw- / A2 B62 Cw4
Shared HLA Haplotypes
Ferritin 2
Biopsy-proven Hemochromatosis
2 1 1
1761 790 54 69
Yes Yes No No
The proband (WL) was diagnosed and this study conducted in 1982, at LSU Medical Center. normal range is 10-287 nanograms/ml; values for proband WL and BS were obtained at diagnosis.
2 Ferritin
48
January 1991 Volume 301 Number 1
Bryan
example was the effect of iron on the mixed lymphocyte culture (MLC) response, which measures the proliferative capacity of CD4 helper T cells in response to disparate HLA Class II antigens. Iron inhibited the proliferative response differentially with respect to HLA Class I phenotype in that the MLC response of individuals who possessed HLA,-A2 was resistant to the inhibitory influence of iron, whereas, inhibition was evident in the MLC response of those who lacked HLA-A2.6 A vast body of literature has accumulated regarding the relationship of iron with susceptibility to infections, and inflammatory and neoplastic diseases. It seems likely that the ability of microorganisms to compete successfully with the host for iron is a feature ofpathogenicity or virulence, and the ability of the host to limit availability of iron to the pathogens is associated with resistance to infection. In view of the potential in vivo immunoregulatory effects of iron,4-s we have evaluated patients with hereditary hemochromatosis to determine if quantitative or functional components of their immune system differed with respect to iron status. 7•S At least two observations in one such studys were consistent with but do not prove the postulate, that the elevated level of storage iron has an in vivo immunoregulatory influence. First, the absolute number of CDS positive T cells were significantly elevated in untreated hereditary hemochromatosis patients and reduced in treated patients, as compared with controls. Second, the proliferative response of peripheral blood mononuclear cells from untreated hereditary hemochromatosis patients to mitogens and in the mixed lymphocyte culture was suboptimal, whereas the response of peripheral blood mononuclear cells from treated hereditary hemochromatosis patients was normal. Immunoglobulin secretion by peripheral blood mononuclear cells from treated hereditary hemochromatosis patients was altered as compared to controls.s Finally, increased expression of the T cell surface receptor that forms thermostable erythrocyte-rosettes (TE-R) was noted. The TE-R receptor is normally expressed on thymocytes or activated T cells and not mature T cells. There was no association of TE-R receptor expression with iron status. 7 Taken together, these data define specific immune alterations in patients with hereditary hemochromatosis with certainty, consistent with the interpretation that cellular immunity may be influenced by the high level of storage iron in patients with hereditary hemochromatosis. Summary
In view of the incidence of hereditary hemochromatosis being close to one individual per 500 and the observation that the incidence of liver cancer in patients with HH is 219 times that of control populations,9 the diagnosis of
THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
HH and its subsequent treatment in the pre-cirrhotic stage is imperative. Although the gene(s) responsible for development of HH is unknown, the hemochromatosis gene is known to be recessive and linked to the HLA gene complex. Therefore, HLA genotyping in families where HH has been diagnosed to determine the linear arrangement of the HLA antigens in the proband may be an appropriate diagnostic adjunct in an effort to identify siblings who are in the pre-cirrhotic stage of hereditary hemachromatosis. Although a milange of immunologic abnormalities have been documented in patients with iron deficiency and in iron overload states, the clinical importance of such abnormalities remains largely undefined. Despite the less clear, clinically important immunologic consequences of iron overload, the immunoregulatory role of iron and its binding proteins clearly has been established and in the future should provide further important insights into the function of the immune system. One of many possible mechanisms that may, in part, explain the high incidence of liver cancer in hereditary hemochromatosis may be related to an effect of iron on the immunosurveillance network, such that in certain clinical situations of high body-iron storage, the immunoregulatory balance may be modified to create an environment that is more conducive to the growth and development of cancer cells. We are in the process of evaluating that postulate. References 1. Edwards CQ, Skolnick MH, Kushner JP: Hereditary hemochromatosis contributions of genetic analyses. Prog Bemat 7: 43-71, 1981. 2. Simon M, Bourel M, Fauchet R, Genetet B: Association of HLAA3 and HLA-BI4 antigens with idiopathic hemochromatosis. Gut 17:332-334, 1976. 3. Edwards CQ, Griffen LM, Dadone MM, Skolnick MH, Kushner JP: Mapping the locus for hereditary hemochromatosis: Localization between HLA-B and HLA-A. Am J Bum Genet 38:805811,1986. 4. de Sousa M, NishiyaK: Inhibition of E-Rosette formation by two iron salts. Cell Immunol38:203-208, 1978. 5. Bryan CF, Leech SH, Bozelka B: The immunoregulatory nature of iron. II. Lymphocyte surface marker expression. J Leukoc Biol 40:589-600, 1986. 6. Bryan CF, Nishiya K, Pollack MS, Dupont B, de Sousa M: Differential inhibition of the MLR by iron: Association with HLA phenotype. Immunogenetics 12:129-140, 1981. 7. Bryan CF, Leech SH, Ducos R, Edwards CQ, KushnerJP, Skolnick MH, Bozelka B, Linn JC, Gaumer R: Thermostable erythrocyte rosette-identification in peripheral blood. J Clin Immunol 4:2,1984. 8. Bryan CF, Leech SH, Kumar P, Gaumer R, Bozelka B, Morgan J: The immune system in hereditary hemochromatosis: A quantitative and functional assessment of the cellular arm. Am J Med Sci 301(1):55-61, 1991. 9. Niederau C, Fischer R, Sonnenberg A, Stremmel W, Trampisch HJ, Strohmeyer G: Survival and causes of death in cirrhotic and in noncirrhotic patients with primary hemochromatosis. N Engl J Med 313:1256-1262, 1985.
49
