High prevalence of abnormal glucose homeostasis secondary to decreased insulin secretion in individuals with hereditary haemochromatosis.

AIMS/HYPOTHESIS: The prevalence and mechanisms of diabetes in hereditary haemochromatosis are not known. We therefore measured glucose tolerance, insulin secretory capacity and insulin sensitivity in adults with haemochromatosis. SUBJECTS AND METHODS: Subjects recruited from referrals to a haemochromatosis clinic underwent OGTT and frequently sampled IVGTT. A chart review of former clinic patients was also performed. RESULTS: The prevalence of diabetes (23%) and IGT (30%) was increased in haemochromatosis compared with matched control subjects (0% diabetes and 14% IGT). Subjects with haemochromatosis and diabetes were overweight (14%) or obese (86%). The prevalence of diabetes, as determined by chart review of fasting glucose values, in subjects who had haemochromatosis and were in the 40-79 years age range was 26%. Overall, patients with haemochromatosis and control subjects had similar values for acute insulin response to glucose and insulin sensitivity. However, patients with haemochromatosis and IGT had a 68% decrease in acute insulin response to glucose (p<0.02) compared with those with NGT. They were not insulin-resistant, exhibiting instead a 62% increase in insulin sensitivity (NS). Haemochromatosis subjects with diabetes exhibited further declines in acute insulin response to glucose, insulin resistance, or both. CONCLUSIONS/INTERPRETATION: Diabetes and IGT are common in haemochromatosis, justifying screening for diabetes and therapeutic phlebotomy. The major abnormality associated with IGT is decreased insulin secretory capacity. Diabetes is usually associated with obesity and concomitant insulin resistance.

Preserving the national blood supply.

This paper examines the current state of the blood supply in the US and focuses on the potential for augmenting blood availability by attention to the iron status of donors. Increasing demands are being made upon the national blood supply as rates of blood donation are declining, in part because of the loss of blood donors as a result of enhanced screening and testing procedures. Iron-related means of expanding the blood supply include the use of blood from individuals undergoing therapeutic phlebotomy for hereditary hemochromatosis and enhancing the retention and commitment of women of childbearing age as donors by using iron supplementation to prevent iron deficiency. In Section I, Dr. Klein discuss the circumstances responsible for a decline in the population of eligible donors, including public attitudes toward donation, factors influencing the retention of donors by blood centers, and the effects of increased screening and testing to maintain the safety of the blood supply. In Section II, Drs. Kushner and Ajioka focus on the consequences of the decision by the US Food and Drug Administration (FDA) to develop recommendations to permit blood centers to collect blood from patients with hereditary hemochromatosis and to distribute this blood obtained without disease labeling if all other screening and testing procedures are passed. After summarizing the pathophysiology of hereditary hemochromatosis, the use by blood centers of blood obtained from heterozygotes and homozygotes for hereditary hemochromatosis is considered. In Section III, Dr. Brittenham reviews the use of low dose, short-term carbonyl iron supplementation for women donors of childbearing age. Replacing the iron lost at donation can help prevent iron deficiency in women of childbearing age and, by decreasing deferral, enhance the retention and commitment of women who give blood regularly. He emphasizes the use by blood centers of iron-related means to enhance recruitment and retention of blood donors.

Transdermal estrogen replacement therapy in postmenopausal women previously treated for porphyria cutanea tarda.

Oral contraceptives and postmenopausal estrogen replacement therapy are recognized as risk factors for the development of porphyria cutanea tarda (PCT) in women. The recommended clinical practice is to withhold estrogen therapy in women who have had phlebotomy therapy for PCT and are clinically and biochemically normal. We tested the safety and efficacy of transdermal estrogen replacement therapy in 7 women previously treated for PCT and compared them with 19 non-porphyric control subjects treated with transdermal or oral estrogens. Gonadotrophic hormone levels, estrogen levels, liver function studies, body iron stores, urine porphyrin excretion, and cytochrome P4501A2 (CYP1A2) activity were monitored for 1 year. Four of the women previously treated for PCT completed the study. None had evidence of a porphyric relapse. CYP1A2 activity, measured by three different methods, did not differ between study subjects receiving estrogens, patients with active PCT, and non-porphyric control subjects, nor did CYP1A2 activity change during the study period. Gonadotrophic hormone levels fell and estrogen levels rose in all women receiving estrogens. The administration of estrogens by the transdermal route appeared to be safe in the small number of subjects we studied and should be considered for women previously treated for PCT.

Disease-related conditions in relatives of patients with hemochromatosis.

BACKGROUND: Hemochromatosis occurs in approximately 5 white people per 1000 and is usually due to homozygosity for mutations in the HLA-linked HFE gene. Although screening has been proposed, the proportion of homozygotes with conditions related to hemochromatosis is uncertain. METHODS: We studied the prevalence of disease-related conditions among relatives of probands with hemochromatosis. We identified probands who presented to a clinic with signs or symptoms of hemochromatosis or who had elevated transferrin-saturation values. We identified homozygous relatives, mainly siblings, on the basis of HLA identity with the proband and by HFE genotyping. Disease-related conditions were cirrhosis, hepatic fibrosis, elevated amino-transferase values, and hemochromatotic arthropathy. RESULTS: We identified 214 homozygous relatives of 291 homozygous probands. Of the 113 men in this group (mean age, 41 years), 96 (85 percent) had iron overload, and 43 (38 percent) had at least one disease-related condition. Of the 52 men over 40 years of age, 27 (52 percent) had at least one disease-related condition. Of the 101 female homozygous relatives (mean age, 44 years), 69 (68 percent) had iron overload, and 10 (10 percent) had at least one disease-related condition. Of the 43 women over 50 years of age, 7 (16 percent) had at least one disease-related condition. If the proband had a disease-related condition, relatives who were men were more likely to have morbidity than if the proband had no disease-related condition. CONCLUSIONS: A substantial number of homozygous relatives of patients with hemochromatosis--more commonly men than women--have conditions related to hemochromatosis that have yet to be detected clinically.

Hemochromatosis genes and other factors contributing to the pathogenesis of porphyria cutanea tarda.

Inherited and acquired factors have been implicated in the pathogenesis of porphyria cutanea tarda (PCT), a disorder characterized by a photosensitive dermatosis and hepatic siderosis. This study, comprising 108 patients with PCT, was intended to define the role of hemochromatosis gene (HFE) mutations in the expression of PCT and to determine the contribution of acquired factors including alcohol, hepatitis C virus (HCV), and estrogen. The 2 known HFE mutations, cysteine 282 tyrosine (Cys282Tyr) and histidine 63 asparagine (His63Asp), were detected by polymerase chain reaction, and anti-HCV immunoglobulin G was detected serologically. Liver biopsies were graded for iron content, inflammation, and fibrosis. Estimates of alcohol and estrogen use were based on a questionnaire. Of the PCT patients tested, 19% were homozygous for the Cys282Tyr mutation; controls were equal to 0.5%. The compound heterozygous genotype was detected in 7% of the PCT patients; controls were less than 1%. The transferrin saturation, serum ferritin, and liver iron burden of all PCT patients were higher than those of nonporphyric controls. The highest values were found in PCT patients homozygous for the Cys282Tyr mutation. Of the patients studied, 59% were HCV positive (compared with 1.8% of the population), and 46% consumed more than 70 g of alcohol daily. Of the female patients, 63% were ingesting estrogens. Hepatic damage was most marked in patients with the Cys282Tyr/Cys282Tyr genotype who had HCV and drank heavily. Homozygosity for the Cys282Tyr mutation and HCV are the greatest risk factors for expression of PCT, and in most patients, more than 1 risk factor was identified. It was common for patients with HCV to consume alcohol. Patients with PCT should be screened for HFE mutations and for HCV. (Blood. 2000;95:1565-1571)

Homogeneous multiplex genotyping of hemochromatosis mutations with fluorescent hybridization probes.

Multiplex polymerase chain reaction amplification and genotyping by fluorescent probe melting temperature (Tm) was used to simultaneously detect multiple variants in the hereditary hemochromatosis gene. Homogenous real-time analysis by fluorescent melting curves has previously been used to genotype single base mismatches; however, the current method introduces a new probe design for fluorescence resonance energy transfer and demonstrates allele multiplexing by Tm for the first time. The new probe design uses a 3'-fluorescein-labeled probe and a 5'-Cy5-labeled probe that are in fluorescence energy transfer when hybridized to the same strand internal to an unlabeled primer set. Two hundred and fifty samples were genotyped for the C282Y and H63D hemochromatosis causing mutations by fluorescent melting curves. Multiplexing was performed by including two primer sets and two probe sets in a single tube. In clinically defined groups of 117 patients and 56 controls, the C282Y mutation was found in 87% (204/234) of patient chromosomes, and the relative penetrance of the H63D mutation was 2.4% of the homozygous C282Y mutation. Results were confirmed by restriction enzyme digestion and agarose gel electrophoresis. In addition, the probe covering the H63D mutation unexpectedly identified the A193T polymorphism in some samples. This method is amenable to multiplexing and has promise for scanning unknown mutations.

Allelic association under map error and recombinational heterogeneity: a tale of two sites.

Recombination acts on the genetic map, not on the physical map. On the other hand, the physical map is usually more accurate. Choice of the genetic or physical map for positional cloning by allelic association depends on the goodness of fit of data to each map under an established model. Huntington disease illustrates the usual case in which the greater reliability of physical data outweighs recombinational heterogeneity. Hemochromatosis represents an exceptional case in which unrecognized recombinational heterogeneity retarded positional cloning for a decade. The Malecot model performs well for major genes, but no approach assuming either equilibrium or disequilibrium has been validated for oligogenes contributing to common disease. In this case of greatest interest, the power of allelic association relative to linkage is less clear than for major genes.

Screening for hemochromatosis: phenotype versus genotype.

Hereditary hemochromatosis is one of the most common inherited disorders among Caucasians of European ancestry. Malregulation of iron absorption from the duodenum eventually leads to iron overload. Although the time required to become iron loaded is variable, it is clear that most homozygotes will eventually become symptomatic. The clinical manifestations can be prevented by prophylactic phlebotomy therapy. Screening young populations is therefore a key to the prevention of disease-related morbidity. Protocols based on the phenotype of high transferrin saturation already exist. The recent identification of a candidate gene for hemochromatosis now allows for a potential genetic screen. Both the phenotypic and the genotypic methods of screening have inherent advantages and disadvantages. Iron-depletion therapy of homozygotes before the development of disease-related morbidity results in normal longevity. National initiatives for hemochromatosis screening will prevent morbidity by identifying and treating young, healthy homozygotes. Healthy, iron-depleted homozygotes should be eligible for health and life insurance at standard rates. Furthermore, healthy homozygotes would make ideal blood donors.

Haplotype analysis of hemochromatosis: evaluation of different linkage-disequilibrium approaches and evolution of disease chromosomes.

We applied several types of linkage-disequilibrium calculations to analyze the hereditary hemochromatosis (hh) locus. Twenty-four polymorphic markers in the major histocompatibility complex (MHC) class I region were used to haplotype hh and normal chromosomes. A total of 169 hh and 161 normal chromosomes were analyzed. Disequilibrium values were found to be high over an unusually large region beginning 150 kb centromeric of HLA-A and extending nearly 5 Mb telomeric of it. Recombination in this region was approximately 28% of the expected value. This low level of recombination contributes to the unusually broad region of linkage disequilibrium found with hh. The strongest disequilibrium was found at locus HLA-H (delta = .84) and at locus D6S2239 (delta = .85), a marker approximately 10 kb telomeric to HLA-H. All disequilibrium methods employed in this study found peak disequilibrium at HLA-H or D6S2239. The cys282tyr mutation in HLA-H, a candidate gene for hh, was found in 85% of disease chromosomes. A haplotype phylogeny for hh chromosomes was constructed and suggests that the mutation associated with the most common haplotype occurred relatively recently. The age of the hh mutation was estimated to be approximately 60-70 generations. Disequilibrium was maintained over a greater distance for hh-carrying chromosomes, consistent with a recent mutation for hh. Our data provide a reasonable explanation for previous difficulties in localizing the hh locus and provide an evolutionary history for disease chromosomes.

Recombinations defining centromeric and telomeric borders for the hereditary haemochromatosis locus.

Hereditary haemochromatosis (HFE) is a common inherited disorder, affecting approximately five per thousand white people of northern European descent. Genetic linkage and linkage disequilibrium studies indicate that the disease locus is tightly linked to HLA-A and D6S105. Recombination between HFE and HLA class I loci is known to be rare. We report here two pedigrees in which recombinations telomeric of HLA-A occurred. These recombinant events define new centromeric and telomeric borders for the HFE locus.

The late chlamydial inclusion membrane is not derived from the endocytic pathway and is relatively deficient in host proteins.

Chlamydiae are obligate intracellular parasites which multiply within infected cells in a membrane-bound structure termed an inclusion. Newly internalized bacteria are surrounded by host plasma membrane; however, the source of membrane for the expansion of the inclusion is unknown. To determine if the membrane for the mature inclusion was derived by fusion with cellular organelles, we stained infected cells with fluorescent or electron-dense markers specific for organelles and examined inclusions for those markers. We observed no evidence for the presence of endoplasmic reticulum, Golgi, late endosomal, or lysosomal proteins in the inclusion. These data suggest that the expansion of the inclusion membrane, beginning 24 h postinoculation, does not occur by the addition of host proteins resulting from either de novo host synthesis or by fusion with preexisting membranes. To determine the source of the expanding inclusion membrane, antibodies were produced against isolated membranes from Chlamydia-infected mouse cells. The antibodies were demonstrated to be solely against Chlamydia-specified proteins by both immunoprecipitation of [35S]methionine-labeled extracts and Western blotting (immunoblotting). Techniques were used to semipermeabilize Chlamydia-infected cells without disrupting the permeability of the inclusion, allowing antibodies access to the outer surface of the inclusion membrane. Immunofluorescent staining demonstrated a ring-like fluorescence around inclusions in semipermeabilized cells, whereas Triton X-100-permeabilized cells showed staining throughout the inclusion. These studies demonstrate that the inclusion membrane is made up, in part, of Chlamydia-specified proteins and not of existing host membrane proteins.

A transcription map of the major histocompatibility complex (MHC) class I region.

We have applied cDNA hybridization selection to nine YACs spanning 3 Mb of genomic DNA from a region centromeric to HLA-A to the histone cluster that lies telomeric to the human major histocompatibility complex (MHC). In addition to Class I genes and pseudogenes, we describe over 63 genes and 23 additional expressed sequence tags distributed throughout the region. Many of the full-length genes belong to gene families. Prominent among these are a group of genes encoding proteins showing homology to the carboxyl-terminal sequences of butyrophilin and an additional group of zinc finger genes. We also detected several previously undefined genes that are specifically expressed in cells of the immune system, indicating a more complex role of the MHC in the immune response than has been appreciated.

The opaH locus of Neisseria gonorrhoeae MS11A is involved in epithelial cell invasion.

In order to produce a successful infection, Neisseria gonorrhoeae (GC) must attach to and invade mucosal epithelial cells. To identify GC gene products involved in this early interaction with host cells we constructed a gene bank derived from a clinical isolate of GC, and isolated a clone which had the capacity to adhere to the human endometrial adenocarcinoma tissue-culture line HEC-1-B. The cloned sequence was identified as a member of the opa gene family whose protein products have been associated with virulence. The GC chromosome contains numerous variant opa genes which, in MS11, are designated opaA-K. Previous work showed that expression of opaC confers a highly invasive phenotype upon strain MS11. When our cloned opa gene was mutated and returned to the GC MS11A chromosome by transformation and homologous recombination, we isolated one transformant that was significantly reduced in its invasive capacity. The locus mutated in this transformant was identified as opaH. Our results indicate that invasiveness of GC for human epithelial cells can be determined by more than one opa gene in strain MS11A.

Mapping recombinant events with molecular markers in hemochromatosis pedigrees.

The gene responsible for hereditary hemochromatosis (HH) is tightly linked to the class I region of the human leukocyte antigen (HLA) complex. Initial studies designed to map the disease locus have relied on serological markers for the class I antigens. Molecular markers from this region can now be used in combination with HLA serotyping for mapping studies. We previously reported two pedigrees in which serological data indicated recombinant events within the class I region. These data suggested a location for the HH locus between HLA-A and HLA-B. Molecular mapping studies have allowed us to demonstrate that an apparent recombination in one pedigree did not occur. This approach has also produced a more precise centromeric boundary for the region containing the disease locus, telomeric of HLA-C. These results emphasize the importance of including both serological and molecular markers in pedigree studies aimed at fine mapping the HH locus.

Shuttle mutagenesis of Neisseria gonorrhoeae: pilin null mutations lower DNA transformation competence.

The method of shuttle mutagenesis has been extended to Neisseria gonorrhoeae. We have constructed a defective mini-Tn3 derivative that encodes chloramphenicol resistance in both N. gonorrhoeae and Escherichia coli and selected for mutations in the chloramphenicol resistance gene that express higher levels of antibiotic resistance in N. gonorrhoeae. Isogenic N. gonorrhoeae strains that differ only in pilin expression were constructed and used to test the effect of pilin null mutations on DNA transformation competence.

Identification and characterization of a Neisseria gonorrhoeae gene encoding a glycolipid-binding adhesin.

We recently identified a set of mammalian cell receptors for Neisseria gonorrhoeae that are glycolipids. These receptors, lactosylceramide [Gal(beta 1-4)Glc(beta 1-1)Cer], gangliotriosylceramide [GalNAc( beta 1-4)Gal(beta 1-4)Glc(beta 1-1)Cer], and gangliotetraosylceramide [Gal(beta 1-3)GalNAc(beta 1-4)Gal(beta 1-4)Glc(beta 1-1)Cer], were shown to be specifically bound by a gonococcal outer membrane protein distinct from pilin and protein II. Here we report the isolation of the gene encoding the gangliotetraosylceramide-binding adhesin from a N. gonorrhoeae MS11 gene bank in Escherichia coli. Transposon mutagenesis studies in E. coli indicate that the adhesion is a protein with a molecular mass of 36,000 Da. The gene encoding the 36-kDa protein is duplicated in MS11 since two transposon insertions were required to abolish expression of the gene in this bacterium. This protein is present on the surface of the gonococcus and is not associated with the pilus.

Cohort movement of different ligands and receptors in the intracellular endocytic pathway of alveolar macrophages.

The rate of movement of different receptors and ligands through the intracellular endocytic apparatus was studied in alveolar macrophages. Cells were exposed to iodinated alpha-macroglobulin-protease complexes, mannose terminal glycoproteins, diferric transferrin, and maleylated proteins. By use of the diaminobenzidine density shift procedure, we demonstrated that these ligands were internalized into the same endocytic vesicle. We then compared the rates of transfer to the lysosome or recycling to the cell surface of different ligands/receptors contained in the same endosome. We found that although the rate constant for degradation was ligand specific, the lag time prior to the initiation of degradation was the same for all three ligands. We also found that molecules taken up nonspecifically by fluid-phase pinocytosis had the same lag time prior to degradation as ligands internalized via receptor-mediated endocytosis. These data suggest that different molecules within the same endocytic compartment are transferred to the lysosome (or degradative compartment) at the same rate. We measured the rate of return of receptors to the cell surface by either inactivating surface receptors by protease treatment at 0 degrees C, or by incubating cells with saturating amounts of nonradioactive ligand at 37 degrees C. We then measured the rate of appearance of "new" receptors on the cell surface. Using these approaches, we found that three different receptors were transferred from internal pools to the cell surface at the same rate. The rate of transfer was independent of whether receptors were initially occupied or unoccupied. Our observations indicate that receptor/ligands, once inside alveolar macrophages, are transported by vesicles which transfer their contents as a cohort from one compartment to another. The rate of movement of these receptors is determined by the movement of vesicles and is independent of their content.

DNA transformation leads to pilin antigenic variation in Neisseria gonorrhoeae.

Many pathogenic bacteria express pili (fimbriae) on their cell surfaces. These structures mediate binding of bacteria to host tissues, and may also be involved in other aspects of pathogenesis. Neisseria gonorrhoeae pili are mainly composed of a single protein, pilin, whose expression is controlled at chromosomal expression loci (pilE). An intact pilin gene and promoter sequences are only found at pilE. Strain MS11 contains two expression sites (pilE1 and pilE2), whereas several of its derivatives and other clinical isolates contain only one. Silent pilin loci (pilS1-pilS7) contain truncated variant pilin genes lacking the promoter and conserved pilin gene sequences. Pilin antigenic variation in N. gonorrhoeae occurs by DNA recombination between one of he silent partial variant gene segments in pilS and an expressed pilin gene in pilE. The recombination reactions are nonreciprocal, and therefore the mechanism has been classified as gene conversion. We report that much of the recombination between pilin loci actually occurs after transformation of living piliated cells by DNA liberated from lysed cells within a population. This constitutes a new molecular mechanism for an antigenic variation system, as well as the first specific function for a DNA transformation system.

Alternative model for Neisseria gonorrhoeae pilin variation.

The pilus of Neisseria gonorrhoeae, a dominant outer membrane organelle, is a major virulence factor. The pilus undergoes phase variation and antigenic variation has also been observed, both in vitro and in vivo. The current model of pilus variation invokes a gene conversion type recombination between a silent pilin locus and the pilin expression site. Experimental results which led to the creation of this hypothesis are reviewed and data are presented which support an alternative model based on DNA transformation.

Characterization of endocytic compartments using the horseradish peroxidase-diaminobenzidine density shift technique.

We have employed a modification of the horseradish peroxidase (HRP)-diaminobenzidine density shift technique of Courtoy et al. (J. Cell Biol., 1984, 98:870-876) to examine the biochemical properties of the endosome. This organelle is involved in receptor recycling and the sorting of internalized receptor ligand complexes. Transferrin covalently bound to HRP was used to place peroxidase activity specifically within the endosome. The peroxidase-catalyzed polymerization of diaminobenzidine within these vesicles causes an increase in buoyant density, thus allowing them to be separated from other membranes. Using this technique we demonstrate that 125I-low density lipoprotein, 131I-epidermal growth factor, and Tf-HRP are internalized into the same endosome. We discovered that the diaminobenzidine reaction product "cross-links" the lumen of the vesicle, rendering vesicular components detergent insoluble. Furthermore, the reaction inactivates enzymatic activities associated with the endosome. Thus, the diaminobenzidine density shift procedure has limited usefulness in studies designed to isolate endosomal constituents. Nonetheless, we have found that the inactivation of enzymatic activities is confined to those endosomes that contain peroxidase. This selectivity allows us to define endosome-specific activities.