Cytokine and Gene Expression Profiling in Patients with HFE-Associated Hereditary Hemochromatosis according to Genetic Profile

Background: Hemochromatosis gene (HFE)-associated hereditary hemochromatosis (HH) is characterized by downregulation of hepcidin synthesis, leading to increased intestinal iron absorption. Objectives: The objectives were to characterize and elucidate a possible association between gene expression profile, hepcidin levels, disease severity, and markers of inflammation in HFE-associated HH patients. Methods: Thirty-nine HFE-associated HH patients were recruited and assigned to 2 groups according to genetic profile: C282Y homozygotes in 1 group and patients with H63D, as homozygote or in combination with C282Y, in the other group. Eleven healthy first-time blood donors were recruited as controls. Gene expression was characterized from peripheral blood cells, and inflammatory cytokines and hepcidin-25 isoform were quantified in serum. Biochemical disease characteristics were recorded. Results: Elevated levels of interleukin 8 were observed in a significant higher proportion of patients than controls. In addition, compared to controls, gene expression of ζ-globin was significantly increased among C282Y homozygote patients, while gene expression of matrix metalloproteinase 8, and other neutrophil-secreted proteins, was significantly upregulated in patients with H63D. Conclusion: Different disease signatures may characterize HH patients according to their HFE genetic profile. Studies on larger populations, including analyses at protein level, are necessary to confirm these findings.

Retrospective Evaluation of Patients Referred for Hemochromatosis Genetic Testing

Background: The common genetic test for hemochromatosis involves the genotyping of two polymorphisms in the HFE gene (C282Y and H63D). Current guidelines suggest testing of patients with ferritin greater than 300 µg/L and transferrin saturation greater than 45%. Testing of first degree relatives of confirmed HFE positive cases is also endorsed. However, the current test has poor sensitivity and specificity for iron overload causing end organ damage. We sought to evaluate biochemical parameters of patients referred for HFEtesting and identify those who received phlebotomy. Methods: After receiving ethics approval, electronic charts of patients referred to Hamilton Health Sciences, Hamilton, Ontario, Canada for HFE genetic testing between January 1, 2012 and December 31, 2012 were reviewed. Results: HFE genetic testing in 642 patients yielded 100 (15.6%) positive C282Y homozygote or C282Y/H63D compound heterozygote results. In patients with biochemical markers of iron overload available (n=160), patients with a risk HFE genotype had significantly higher iron saturation, serum iron, and hemoglobin (P<0.001), but did not have higher ferritin or liver enzymes. Only fifty percent of patients referred had biochemical evidence of iron overload (transferrin saturation [TS] > 45% and ferritin > 300). Of patients who required phlebotomy, only 27 of 40 (67.5%) were HFEpositive. Conclusion: Many factors affect the decision to phlebotomize a patient with suspected iron overload. However, it appears that most of the current HFE genetic testing being performed did not alter patient management. Disclosures Crowther: Asahi Kasai: Membership on an entity's Board of Directors or advisory committees; Bayer: Speakers Bureau; Celgene: Speakers Bureau; Shire: Speakers Bureau; Boehriniger Ingelheim: Consultancy; CSL Behring: Speakers Bureau; Leo Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; Portola: Membership on an entity's Board of Directors or advisory committees; Viropharma: Membership on an entity's Board of Directors or advisory committees.

Three Kinships with ALAS2 P520L Mutations, Two in Association with Severe Iron Overload, and One with Sideroblastic Anemia and Severe Iron Overload.

Aminolevulinate synthase 2 (ALAS2) is an erythroid-expressed gene located on chromosome Xp11.21. Mutations in ALAS2 have been shown to be associated with sideroblastic anemia. We have identified a novel mutation in ALAS2, P520L, in three kinships. The P520L mutation was not found in 316 white male control subjects. The proline in this position is highly conserved across species from humans to zebrafish. In the C kinship, the P520L mutation was first identified in a white man who presented with severe iron overload at an early age and was a HFE C282Y homozygote. Genetic analyses of members of the C kinship suggest that the presence of P520L alone in hemizygous males, or simple heterozygosity in females, is not associated with anemia or iron overload. Females heterozygous for both HFE C282Y and ALAS2 P520L also had normal ferritin levels. Only subjects homozygous for HFE C282Y and hemizygous or heterozygous for ALAS2 P520L had severe iron overload. Sequencing studies revealed that the propositus did not have missense mutations in HAMP, HJV, FPN1, ABC7, IL6, or RAG1. In the H kinship, the propositus was a white woman with severe iron overload who was heterozygous for ALAS2 P520L; she had a wildtype HFE genotype. The pedigree of the H kinship identified 5 additional females who were heterozygous for the P520L ALAS2 mutation. Only the propositus had iron overload and none of the subjects with P520L had sideroblastic anemia. The propositus did not have missense mutations in FPN1, HAMP, HJV, TFR2, B2M, IRP2, ABC7, or SFT. We speculate that the propositus in the H kinship has a mutation in a currently unknown gene that contributes to her severe iron overload. In the S kinship, the P520L mutation was identified in a white man with sideroblastic anemia and severe iron overload. The patient had a second mutation in ALAS2, R560H, a mutation previously described in two brothers with sideroblastic anemia of variable penetrance (Blood100:4236–4238, 2002). It is not possible to determine the effect of the P520L mutation on the penetrance of the R560H-associated sideroblastic anemia and iron overload. We conclude that the ALAS2 P520L occurs at a very low allele frequency in the white population. The present observations also suggest that there is no distinctive phenotype associated with the P520L mutation alone, but that P520L may act as a modifier of iron overload in the presence of HFE homozygosity, other missense mutations of ALAS2, or mutations of uncharacterized iron regulatory genes.

Four New Mutations in the Erythroid-Specific 5-Aminolevulinate Synthase (ALAS2) Gene Causing X-Linked Sideroblastic Anemia: Increased Pyridoxine Responsiveness After Removal of Iron Overload by Phlebotomy and Coinheritance of Hereditary Hemochromatosis

X-linked sideroblastic anemia (XLSA) in four unrelated male probands was caused by missense mutations in the erythroid-specific 5-aminolevulinate synthase gene (ALAS2). All were new mutations: T647C, C1283T, G1395A, and C1406T predicting amino acid substitutions Y199H, R411C, R448Q, and R452C. All probands were clinically pyridoxine-responsive. The mutation Y199H was shown to be the first de novo XLSA mutation and occurred in a gamete of the proband’s maternal grandfather. There was a significantly higher frequency of coinheritance of the hereditary hemochromatosis (HH)HFE mutant allele C282Y in 18 unrelated XLSA hemizygotes than found in the normal population, indicating a role for coinheritance ofHFE alleles in the expression of this disorder. One proband (Y199H) with severe and early iron loading coinherited HH as a C282Y homozygote. The clinical and hematologic histories of two XLSA probands suggest that iron overload suppresses pyridoxine responsiveness. Notably, reversal of the iron overload in the Y199H proband by phlebotomy resulted in higher hemoglobin concentrations during pyridoxine supplementation. The proband with the R452C mutation was symptom-free on occasional phlebotomy and daily pyridoxine. These studies indicate the value of combined phlebotomy and pyridoxine supplementation in the management of XLSA probands in order to prevent a downward spiral of iron toxicity and refractory anemia.

Four New Mutations in the Erythroid-Specific 5-Aminolevulinate Synthase (ALAS2) Gene Causing X-Linked Sideroblastic Anemia: Increased Pyridoxine Responsiveness After Removal of Iron Overload by Phlebotomy and Coinheritance of Hereditary Hemochromatosis

X-linked sideroblastic anemia (XLSA) in four unrelated male probands was caused by missense mutations in the erythroid-specific 5-aminolevulinate synthase gene (ALAS2). All were new mutations: T647C, C1283T, G1395A, and C1406T predicting amino acid substitutions Y199H, R411C, R448Q, and R452C. All probands were clinically pyridoxine-responsive. The mutation Y199H was shown to be the first de novo XLSA mutation and occurred in a gamete of the proband’s maternal grandfather. There was a significantly higher frequency of coinheritance of the hereditary hemochromatosis (HH)HFE mutant allele C282Y in 18 unrelated XLSA hemizygotes than found in the normal population, indicating a role for coinheritance ofHFE alleles in the expression of this disorder. One proband (Y199H) with severe and early iron loading coinherited HH as a C282Y homozygote. The clinical and hematologic histories of two XLSA probands suggest that iron overload suppresses pyridoxine responsiveness. Notably, reversal of the iron overload in the Y199H proband by phlebotomy resulted in higher hemoglobin concentrations during pyridoxine supplementation. The proband with the R452C mutation was symptom-free on occasional phlebotomy and daily pyridoxine. These studies indicate the value of combined phlebotomy and pyridoxine supplementation in the management of XLSA probands in order to prevent a downward spiral of iron toxicity and refractory anemia.