Severity of iron overload in hemochromatosis: effect of volunteer blood donation before diagnosis.

BACKGROUND: An effort was made to determine if volunteer blood donation before diagnosis decreases the severity of iron overload at diagnosis in persons with hemochromatosis. STUDY DESIGN AND METHODS: A study was performed in 1089 persons in the United States with hemochromatosis who responded to a convenience sample survey and in 124 C282Y/C282Y hemochromatosis probands diagnosed during routine medical care. RESULTS: Less than half of questionnaire respondents (46.2%) and probands (35.5%) reported that they had been volunteer blood donors; 5.4 percent and 4.0 percent, respectively, had donated >20 units of blood. In either subject group, there were no significant differences according to age in the mean numbers of units that needed to be removed by therapeutic phlebotomy to induce iron depletion in subgroups of men and women, respectively. Similarly, there was no significant correlation of units of voluntary blood donation or of therapeutic phlebotomy index (= therapeutic phlebotomy units/age in years) with the number of therapeutic phlebotomy units needed to induce iron depletion. When questionnaire respondents were stratified by sex, there was no significant correlation of units of blood donation with the number of therapeutic phlebotomy units needed to induce iron depletion or with the therapeutic phlebotomy index. CONCLUSION: Routine blood donation does not, on average, decrease the severity of iron overload in persons with hemochromatosis. These findings have implications for the understanding of the severity of iron overload and its complications in hemochromatosis, for advising persons with hemochromatosis about treatment, and for considering persons with hemochromatosis as possible blood donors.

Screening for hemochromatosis in routine medical care: an evaluation of mean corpuscular volume and mean corpuscular hemoglobin.

Our aim was to evaluate the potential utility of mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) to detect hemochromatosis. We computed the accuracy of MCV and MCH cut-off points > or = upper reference limits using data from 94 probands and 132 white controls. Our reference ranges are MCV 80.0-97.0 fL and MCH 26.0-32.0 pg. Sensitivity of MCV was 8.6-48.3% for men and 2.8-44.4% for women (cut-off points > or = 105.0 - > or = 97.0 fL, respectively). Sensitivity of MCH was 33.9-70.7% for men and 19.6-50.0% for women (cut-off points > or = 34.0 - > or = 32.0 pg, respectively). Using MCV and a hemochromatosis frequency typical of many western Caucasian populations (0.005), positive predictive values (PV+) were 2.1-100.0% in men and 4.2-100.0% in women. Using MCH, PV+ were 1.7-8.2% in men and 1.8-6.8% in women. We also calculated PV+ using the hemochromatosis frequency 0.015, which could occur in persons receiving medical care. Using MCV cut-off points > or = 101.0 fL, PV+ were 8.9-100.0% in men and 100.0% in women with maximum sensitivities of 24.1% and 25.0%, respectively. Using MCH testing, PV+ was 21.5% in men (cut-off point > or = 34.0 pg) and 18.2% in women (cut-off point > or = 33.0 pg) with sensitivities of 33.9% and 37.0%, respectively. Using MCV or MCH, sensitivity and PV+ for the HFE genotype C282Y/C282Y were generally greater than for "nonclassical" HFE genotypes. All negative predictive values in our study were > or = 98.5%. We conclude that supranormal values of MCV or MCH could be used to detect hemochromatosis in white persons of western European descent who are receiving routine medical care. Comparisons of MCV, MCH, and transferrin saturation testing and other implications of MCV and MCH testing for hemochromatosis in medical care are discussed.

Peripheral blood erythrocyte parameters in hemochromatosis: evidence for increased erythrocyte hemoglobin content.

We studied peripheral blood erythrocyte parameters and HFE genotypes in 94 hemochromatosis probands and 132 white, normal control subjects. Mean red blood cell counts in probands and control subjects were not significantly different. However, mean values of hemoglobin, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) were significantly higher in C282Y/C282Y probands (n = 60) than in wild-type control subjects (n = 65). Probands with other HFE genotypes also had increased mean erythrocyte parameters (other than red blood cell count). Peripheral blood smears prepared before therapeutic phlebotomy revealed that erythrocytes in many probands had increased diameters and were well filled with hemoglobin. Erythrocyte parameters were similar in C282Y/C282Y probands with and without hepatomegaly, elevated serum concentrations of hepatic enzymes, hepatic cirrhosis, diabetes mellitus, arthropathy, or hypogonadism. Among C282Y/C282Y probands, significantly greater values of MCV (but not other erythrocyte parameters) occurred among those who had transferrin saturation values of 75% or greater or iron overload at diagnosis. After iron depletion, the mean MCV, MCH, and MCHC values of C282Y/C282Y probands decreased but remained significantly greater than values in wild-type control subjects. Mean values of prephlebotomy MCH and MCHC concentrations were lower in HLA-A3-positive than in HLA-A3-negative C282Y/C282Y probands. We conclude that increased values of mean hemoglobin, hematocrit, MCV, MCH, and MCHC in hemochromatosis probands are caused primarily by increased iron uptake and hemoglobin synthesis by immature erythroid cells. Mechanisms of iron uptake by erythrocytes that could explain these results are discussed.

Two novel missense mutations of the HFE gene (I105T and G93R) and identification of the S65C mutation in Alabama hemochromatosis probands.

Sequencing of HFE exons 2, 3, 4, and 5, and of portions of introns 2, 4, and 5 revealed novel mutations in four of twenty hemochromatosis probands who lacked C282Y homozygosity, C282Y/H63D compound heterozygosity, or H63D homozygosity. Probands 1 and 2 were heterozygous for previously undescribed mutations: exon 2, nt 314T-->C (314C; I105T), and exon 2, nt 277G-->(277C; G93R), respectively; these probands were also heterozygous for H63D and C282Y, respectively. Probands 3 and 4 were heterozygous for a previously described but uncommon HFE mutation: exon 2, nt 193A-->T (193T; S65C). Proband 3 was also heterozygous for C282Y and had porphyria cutanea tarda, and Proband 4 had hereditary stomatocytosis. Each of these four probands had iron overload. In each proband with an uncommon HFE coding region mutation, I105T, G93R, and S65C occurred on separate chromosomes from those with the C282Y or H63D mutations. Neither I105T, G93R, nor S65C occurred as spontaneous mutations in our probands. The I105T and G93R mutations were linked to haplotypes bearing HLA-A3,-B7 and HLA-A2,-B62, respectively. The S65C mutation was linked to a haplotype characterized by HLA-32. Sixteen other probands did not have an uncommon HFE exon mutation. In 176 normal control subjects, two were heterozygous for S65C, but I105T and G93R were not detected. Nine of twenty probands were heterozygous and two probands were homozygous for a previously described base-pair change at intron 2, nt 3671T-->C. One proband without a detectable missense mutation had a previously described intron 5 allele (nt 6700G-->A). Heterozygosity for a previously described base-pair change in intron 4 (nt 5636T-->C) was detected in all persons we studied who also had the S65C mutation. One proband was heterozygous for a previously undescribed base-pair change at intron 5 (nt 5807A-->G). We conclude that uncommon HFE exon and intron mutations may be discovered among hemochromatosis patients who have "atypical" HFE genotypes.

Diagnosis of hemochromatosis in family members of probands: a comparison of phenotyping and HFE genotyping.

PURPOSE: We wanted to compare phenotyping and HFE genotyping for diagnosis of hemochromatosis in 150 family members of 61 probands. METHODS: Phenotypes were defined by persistent transferrin saturation elevation, iron overload, or both; genotypes were defined by HFE mutation analysis. RESULTS: Twenty-five family members were C282Y homozygotes; 23 of these (92%) had a hemochromatosis phenotype. Twenty-three family members had HFE genotype C282Y/H63D; eight of these (35%) had a hemochromatosis phenotype. Six of 102 (6%) family members who inherited other HFE genotypes had a hemochromatosis phenotype. CONCLUSION: Phenotyping and genotyping are complementary in diagnosing hemochromatosis among family members of probands.

Genetic and clinical description of hemochromatosis probands and heterozygotes: evidence that multiple genes linked to the major histocompatibility complex are responsible for hemochromatosis.

We evaluated Alabama hemochromatosis probands (n = 74) and normal control subjects (n = 142) for expression of the hemochromatosis-associated mutations nt 845G-->A (845A; Cys282Tyr) and nt 187C-->G (His63Asp) in a gene linked to the major histocompatibility complex (MHC). We also tabulated parameters of iron metabolism and iron overload in probands and in obligate heterozygote family members of homozygous Cys282Tyr probands. Among probands, 59.4% were Cys282Tyr homozygotes and 20.3% were heterozygotes; 20.3% did not express this mutation. In normal control subjects, 14.7% were heterozygous for the Cys282Tyr mutation; one normal control subject was homozygous for the Cys282Tyr mutation. None (0 of 44) of our Cys282Tyr-homozygous hemochromatosis probands had the His63Asp mutation. Of the Cys282Tyr-heterozygous and -negative probands, the His63Asp mutation occurred in 26.7% (4/15) and 53.3% (8/15), respectively. In normal control subjects, 23.2% were heterozygous for the His63Asp mutation; 2.8% were homozygous. Induction phlebotomy requirements and other manifestations of iron overload were significantly greater in Cys282Tyr homozygotes than among other probands. Cys282Tyr-heterozygous probands had significantly higher values of serum iron parameters than did obligate Cys282Tyr heterozygotes whose values were, on the average, normal. Co-expression of HLA-A3, HLA-B7, and D6S105(8) was significantly more frequent in all subgroups of probands stratified by Cys282Tyr expression than in normal control subjects. These results demonstrate that the severity of iron overload in hemochromatosis is affected significantly by genetic factors. Further, our findings support the hypothesis that one or more MHC-linked genes other than that corresponding to the Cys282Tyr and His63Asp mutations contributes to increased iron absorption and iron overload in hemochromatosis probands.

beta2 knockout mice develop parenchymal iron overload: A putative role for class I genes of the major histocompatibility complex in iron metabolism.

Hemochromatosis (HC) is an inherited disorder of iron absorption, mapping within the human major histocompatibility complex (MHC). We have identified a multigene system in the murine MHC that contains excellent candidates for the murine equivalent of the human HC locus and implicate nonclassical class I genes in the control of iron absorption. This gene system is characterized by multiple copies of two head-to-head genes encoded on opposite strands and driven by one common regulatory motif. This regulatory motif has a striking homology to the promoter region of the beta-globin gene, a gene obviously involved in iron metabolism and hence termed beta-globin analogous promoter (betaGAP). Upstream of the betaGAP sequence are nonclassical class I genes. At least one of these nonclassical class I genes, Q2, is expressed in the gastrointestinal tract, the primary site of iron absorption. Also expressed in the gastrointestinal tract and downstream of the betaGAP motif is a second set of putative genes, termed Hephaestus (HEPH). Based on these observations, we hypothesized that the genes that seem to be controlled by the betaGAP regulatory motifs would be responsible for the control of Fe absorption. As a test of this hypothesis, we predicted that mice which have altered expression of class I gene products, the beta2-microglobulin knockout mice, [beta2m(-/-)], would develop Fe overload. This prediction was confirmed, and these results indicate beta2m-associated proteins are involved in the control of intestinal Fe absorption.

Biotinylated diaminopyridine: an approach to tagging oligosaccharides and exploring their biology.

Fluorescent tagging of free oligosaccharides by reductive amination permits sensitive detection and fractionation of these molecules. To expand the scope of this approach, we have synthesized a fluorescent reagent, 2-amino-(6-amidobiotinyl)pyridine. This reagent can tag oligosaccharides under nondegradative conditions with high efficiency. The resulting adducts show excellent fractionation by reverse-phase HPLC with sensitive detection in the low picomole range. When combined with sequential exoglycosidase digestion, stepwise sequencing of the sugar chains is possible. The biotinyl group can also be used to recover the sugar chain from reaction mixtures. The high-affinity interaction of the biotinyl group with multivalent avidin or streptavidin can be used to create the functional equivalent of neoglycoproteins carrying multiple copies of oligosaccharides of defined structure. These complexes allow the production of IgG antibodies directed against the oligosaccharide chain. They can also harness the power of (strept)avidin-biotin technology for the detection and isolation of oligosaccharide-specific receptors from native sources of recombinant libraries.

High efficiency vectors for cosmid microcloning and genomic analysis.

We describe the construction and use of cosmid vectors designed for microcloning, gene isolation and genomic mapping starting from submicrogram amounts of eukaryotic DNA. These vectors contain (1) multiple cos sites to allow for simple and efficient cloning using non size-selected DNA; (2) bacteriophage T3 and T7 promoter sequences flanking the cloning site to allow for the synthesis of end-specific probes for chromosome walking; (3) a selectable gene for immediate gene transfer of cosmid DNA into mammalian cells; (4) recognition sequences for specific oligodeoxyribonucleotides to allow rapid restriction mapping; (5) unique NotI, SacII or SfiI sites flanking the cloning site to allow for removal of the cloned DNA insert from the vector. These cosmid vectors allow the construction of high quality genomic libraries in situations where the quantity of purified DNA is extremely limited, such as when using DNA prepared from purified mammalian chromosomes isolated by fluorescence-activated cell sorting.

Arginine 45 is a major part of the antigenic determinant of human beta 2-microglobulin recognized by mouse monoclonal antibody BBM.1.

Monoclonal antibody BBM.1 (Brodsky, F.M., Bodmer, W. F., and Parham, P. (1979) Eur. J. Immunol. 9, 536-545) identifies an antigenic determinant of human beta 2-microglobulin (beta 2-M). The antibody binds free and HLA-A,B-associated beta 2-M with similar affinity, showing that the BBM.1 antigenic determinant does not involve residues of beta 2-M that interact with HLA-A,B heavy chains. Peptides (SWH.1-5) synthesized from residues 35-50 of the beta 2-M sequence specifically inhibit the binding of BBM.1 to cell surfaces. Their inhibitory activity is destroyed by trypsin treatment. The observations (i) that BBM.1 does not bind to beta 2-M of species other than man, gorilla, and chimpanzee and (ii) that arginine 45 is the only human-specific residue between positions 35 and 50 suggested that this residue might be part of the BBM.1 antigenic determinant. This hypothesis was confirmed by reversible modification of arginine residues with cyclohexanedione. Modification of arginines in native beta 2-M and of the single arginine, corresponding to position 45, in the peptide SWH.5 resulted in up to 95% loss of BBM.1 inhibitory activity. Reversal of the modification by treatment with hydroxylamine resulted in complete recovery of activity. Rabbit antibodies elicited by immunization of SWH.5 conjugated to bovine serum albumin showed no detectable reaction with native beta 2-M but did specifically react with human beta 2-M after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoresis onto nitrocellulose. These results thus identify a region around residue 45 of the beta 2-M polypeptide which is exposed to the environment and not involved in binding HLA-A,B heavy chain. Analysis of the beta 2-M sequence by calculating local hydrophilicity indices (Hopp, T. P., and Woods, K. R. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 3824-3828) agree with this region being a major antigenic determinant. Models of beta 2-M structure as an immunoglobulin domain show this region of polypeptide to be part of a loop between the two layers of beta-pleated sheet, also consistent with it being a major antigenic determinant. The position of the loop favors a model in which beta 2-M interacts with HLA across the four-stranded beta-pleated sheet like an immunoglobulin constant region domain.