Long-term survival of 2 cases of hemochromatosis respectively homozygous for His63Asp and Cys282Tyr mutations]

A 45-year-old Greek patient was found to have a moderate iron overload (ferritin 1213 micrograms/l, serum iron 21.5 mumol/l, transferrin saturation 40%). He underwent 12 phlebotomies of 450 cc over an 8-year period and ferritin was normalized (267 micrograms/l) after the seventh. Study of the HLA-H gene in leukocyte DNA showed that the patient is homozygous for the His63Asp mutation while no modification was found at position 282. This case is compared with that of a 50-year-old Swiss male presenting a severe iron overload (ferritin 7660 micrograms/l, serum iron 36.5 mumol/l, transferrin saturation 97%). Although this patient has undergone 77 phlebotomies (450 cc each time) over a 2-year period, he continues to have a high ferritin level (2200 micrograms/l). HLA-H gene analysis showed the absence of codon 63 mutation and the presence of Cys282Tyr mutation in the homozygous state. The study of these two cases indicates that penetrance of the His63Asp mutation in the homozygous state is very low as compared to Cys282Tyr and results in moderate iron accumulation, probably without organ damage. This genotype must be looked for whenever moderate iron overload is present.     

A PCR-SSP method for detecting the His63Asp mutation in the HFE gene associated with hereditary haemochromatosis

The influence of oral treatment with a suspension of non-pathogenic Escherichia coli cells (commercially available as: Symbioflor II) on the morphological composition of the gut microflora and on the systemic humoral immune response (the IgG-, IgA- and IgM-isotype) against the bacterial cells in the Symbioflor II preparation was measured. After a pretreatment period of 21 days, ten healthy human volunteers ingested 1*10(8) cells of E. coli daily for 14 days. Thereafter a follow-up period of 28 days completed the study. The results of this study indicated that no effect of the treatment on the composition of the gut microflora could be observed. However, the immune-fluorescence measurements revealed a significant increase in circulating amounts of IgG directed against the administered E. coli cells. It is concluded that the treatment only resulted in a specific humoral immune response, while the gut microflora is not modulated.     

Prevalence of the C282Y and H63D mutations in the HFE gene in patients with hereditary haemochromatosis and in control subjects from Northern Germany

Mutation analysis was performed for two HFE mutations (C282Y, H63D) in unrelated patients with hereditary haemochromatosis (n = 92), family members of patients (n = 34), and unrelated controls (n = 157) from Northern Germany, 87/92 patients (94.6%) revealed the C282Y mutation in homozygous form, five were heterozygous. No H63D mutation was found in 174 chromosomes of patients homozygous for C282Y, whereas four of the heterozygote patients also carried the H63D mutation. Among the control group, 9.6% were heterozygotes for C282Y. 2/157 subjects were homozygous, 37/157 were heterozygous for the H63D mutation, but showed no signs of iron overload.     

HFE codon 63/282 (H63D/C282Y) dimorphism in German patients with genetic hemochromatosis

The antinociceptive actions of morphine incorporated into an injectable chitosan-based gel were investigated in rats. Subcutaneous administration of 4.8 mg/kg morphine sulphate in a gel composed of N,O-carboxymethylchitosan (NOCC) and chitosan resulted in significant antinociception within 10 min that was maximal at 60 min and persisted for 6 h. In contrast, the same dose of morphine sulphate injected in sterile saline produced maximal responses at 30 min but only persisted for 2 h. NOCC/chitosan gel was easily injectable using a 22 guage needle and appears stable in long-term storage. No local or systemic adverse effects other than morphine-induced sedation were observed either at the time of injection or during the subsequent 48 h. We conclude that gels composed of chitosan and chitosan derivatives are effective matrices for sustained-release formulations of opioid analgesics capable of providing long-lasting antinociception.     

Rapid diagnosis of asymptomatic hereditary haemochromatosis by detection of the Cys282Tyr mutation in the HLA-H gene

Hereditary haemochromatosis is an autosomal recessive disorder characterised by life-long excessive accumulation of iron. A candidate gene for hereditary haemochromatosis has recently been reported (HLA-H) and a specific missense mutation (Cys282Tyr) has been identified in 85% of patients with the disorder. We describe the rapid detection of this mutation using the polymerase chain reaction and restriction endonuclease digestion. The usefulness of this test for early diagnosis of hereditary haemochromatosis in asymptomatic family members is highlighted.     

Has the hemochromatosis gene been identified? A newly discovered MHC class I gene is mutated in patients with hereditary hemochromatosis

A new method of recruiting people who inject drugs was employed in Adelaide in 1994 to attempt to obtain a response rate and demographic information about those who declined to participate. Numbered invitation cards were distributed to injecting drug users (IDUs). Those choosing not to participate were asked to complete 4 questions on the card and return them. 22.7%. Of all cards distributed resulted in an enquiry about the study and only 1.4% of the cards were returned from those who chose not to participate. This recruiting strategy is as successful as other strategies with the added advantage of estimating response rates.     

Interaction of the hemochromatosis gene product HFE with transferrin receptor modulates cellular iron metabolism

Mutations of a novel MHC class I-like protein, termed HFE, have been found in the vast majority of patients with the iron overload disease heredity hemochromatosis. Identification of HFE is likely to shed light on one of the major enigmas of mammalian iron homeostasis: How is intestinal iron absorption regulated?     

A haplotype and linkage disequilibrium analysis of the hereditary hemochromatosis gene region

Monoclonal antibodies were generated against serotonin (5-HT) and the C-terminal portion of the neuronal form of nitric oxide synthase (nNOS), the enzyme producing nitric oxide in neurons. These antibodies were used to compare the distribution of 5-HT- and nNOS-containing neurons in the raphe nuclei of four animal species (rat, mouse, guinea pig, and cat). It was found that the rat was the only species in which the raphe nuclei contain a substantial number of nNOS-immunoreactive (IR) cell bodies. In this species and as observed by other authors, all mesencephalic raphe nuclei contained nNOS-IR cells, the largest group being located in the nucleus raphe dorsalis. The coexistence of nNOS and 5-HT immunoreactivities in these nuclei was visualized by double labeling. In the medulla, the nuclei raphe magnus and obscurus displayed a rather low number of nNOS-IR neurons. In the other species, nNOS-IR cell bodies were found in very low numbers, whatever raphe nucleus was considered. The rostral pole of the nucleus raphe dorsalis and the nuclei raphe magnus and obscurus contained a few nNOS-IR neurons which did not show any coincidence with the 5-HT neurons. In addition, nNOS-IR axons were rare. It is concluded that in the mouse, guinea pig, and cat the involvement of nitric oxide in functions subserved by 5-HT within the raphe nuclei might be minimal.     

Prevalence of mental retardation in patients with hereditary retinoblastoma

Transvaginal amniotic puncture (TAP) was performed on 20 consecutive missed abortions immediately prior to dilatation and evacuation and the cytogenetic results compared. The information received from products of conception (POC) and TAP was in concordance in only 5 of 20 (25%) cases. Tissue obtained from POC yielded cells in all instances. However, only 3 of 20 POC samples yielded findings other than normal female. In contrast, 92.8% of the conclusive diagnoses would have been achieved by TAP alone. These data strongly suggest that TAP is superior to POC for accurate cytogenetic assessment of missed abortion and should lead to a reevaluation of our current understanding and management of pregnancy loss.     

Patients with type 2 diabetes have a high frequency of the C282Y mutation of the hemochromatosis gene

Although maternal fever has been implicated as a human teratogen in several studies, no prospective study has adequately addressed the full spectrum of birth outcomes following such exposure in pregnancy. The purpose of this study was to determine whether or not maternal fever is associated with an increased risk for structural malformations, prematurity, growth retardation, or pregnancy loss. Using a prospective cohort study design, we ascertained women who had called the California Teratogen Information Service and Clinical Research Program between 1979-1996 with questions regarding fever in a current pregnancy. Of these women, 115 who reported a fever of at least 38.9 degrees C lasting for at least 24 h (high fever group) and 147 women who reported a fever of either less than 38.9 degrees C or lasting less than 24 h (low fever group) were enrolled in the cohort. An additional 298 pregnant women who reported having no fever at any time in pregnancy were enrolled in a control group. All pregnancies were followed in a similar fashion, and outcomes were compared among the three groups. The combined prevalence of all major structural malformations was increased, but not significantly so, in the offspring of women who had a high fever in the first trimester of pregnancy compared to those with a lower fever or to controls (relative risk 1.80 for high fever group compared to controls; 95% confidence interval, 0.54, 6.03; relative risk 1.21 for low fever group compared to controls; 95% confidence interval, 0.36, 4.03). However, 2/34 or 5.9% of women who had a high fever during the critical period for neural tube closure carried fetuses with anencephaly compared to none in the low fever group or controls. Specific minor defects were found more frequently in the high fever group compared to controls and were consistent with the pattern of defects previously reported in a retrospective case series. In addition, stillbirth occurred more frequently in the high fever group compared to controls (2.6% vs. 0%). These data support the conclusion that high maternal fever early in pregnancy is a human teratogen. Women who experience fevers of 38.9 degrees C or higher for extended periods of time in the first month of pregnancy should be considered at increased risk for neural tube defects and should be provided appropriate counseling.     

Wide range of disease onset in a family with Alzheimer disease and a His163Tyr mutation in the presenilin-1 gene

The tetraruthenated porphyrin, mu-[meso-5,10,15,20-tetra(pyridyl)porphyrin]tetrakis[bis-(bipyridine) chloride ruthenium(II)] (TRP) is a supramolecular cationic species. The aim of the present investigation was to evaluate the photodynamic properties of TRP and Zn-TRP to damage DNA with emphasis on the mechanistic aspects. The ability for tetraruthenated porphyrin derivatives to induce photosensitization reactions has been determined using 2'-deoxyguanosine as a DNA model compound. The main photooxidation products of the targeted nucleoside were identified and classified according to their mechanisms of formation, involving either a radical pathway (type I) or a singlet oxygen-mediated mechanism (type II). Quantification of the different oxidation products provides a means to evaluate the relative contribution of type I and type II pathways associated with the oxidative photosensitization of 2'-deoxyguanosine by tetraruthenated porphyrin derivatives. Results indicate that 1O2 plays a major role in the mechanism of photooxidation mediated by these porphyrin derivatives. In addition an increase of the photosensitizing effect in the presence of zinc is observed. For each sensitizer, the ratio between type II and type I photoproducts has been calculated and compared to that of other known dyes such as methylene blue and riboflavin.     

The C282Y mutation in the haemochromatosis gene (HFE) and hepatitis C virus infection are independent cofactors for porphyria cutanea tarda in Australian patients

The interaction of free allergen with two (or more) IgE molecules bound to the high-affinity receptor for IgE (FcepsilonRI) on mast cells and basophilic granulocytes results in the release of inflammatory mediators. The role of allergen-specific IgG antibodies in the allergic reaction in human beings is less clear. We produced two chimeric IgE antibodies, hIgE-Dp2A and hIgE-Dp2B, directed to two nonoverlapping epitopes (A and B) of the house dust mite allergen Der p 2. Chimeric IgG1 and IgG4 variants of these antibodies were produced also. Basophil activation by the house dust mite allergen Der p 2 was induced after sensitization of basophils with a mixture of chimeric hIgE-Dp2A and hIgE-Dp2B antibodies but not after sensitization by the individual IgE antibodies alone. Basophil activation was also shown after sensitization with hIgE-Dp2A and stimulation with Der p 2 incubated with hIgG1-Dp2B or hIgG4-Dp2B antibodies. Both IgE and IgG antibodies directed to the other nonoverlapping epitope complemented the sensitization by the hIgE-Dp2A antibody. Nonsensitized basophils were not activated by the Der p 2/hIgG-Dp2 mixtures. These results indicate that allergen-specific IgG can complement an IgE-dependent reaction and therefore under certain conditions can act as an anaphylactic antibody.     

Microsatellite instability and frameshift mutations in the Bax gene in hereditary nonpolyposis colorectal carcinoma

Patients with ulcerative colitis (UC) are at higher risk for cancer. Risk factors are duration of disease, extent of colitis, associated primary sclerosing cholangitis and possibly early onset of UC in childhood. Epithelial dysplasias are considered as precursors of colorectal cancer within the concept of an inflammation-dysplasia-carcinoma sequence. Dysplasia originates multifocally and is difficult to identify by colonoscopy. Histomorphological diagnosis can also be problematical. Surveillance programs utilize colonoscopy with random biopsies to diagnose dysplasia in patients with risk factors. The efficiency of these programs can be markedly increased when certain rules are applied. The ultimate aim must be to perform a proctocolectomy in patients at higher risk before invasive cancer develops. With only a few exceptions, colorectal cancer in UC can be treated by restorative proctocolectomy. Partial resection of the colon should be avoided because of the high frequency of occult carcinomas and multifocal carcinogenesis. There are first results that indicate a higher risk for malignant deterioration in the terminal ileum. After an ileoanal pouch procedure patients with chronic pouchitis seem to have a higher risk for dysplasia. At the moment the risk for malignancy cannot be calculated because of the relatively short follow-up time after ileoanal pouch procedures. However, it is recommended that after restorative proctocolectomy patients be followed by endoscopy and random biopsies for the rest of their lives.     

Diagnosis of hereditary hemochromatosis with molecular analysis of DNA in patients with anti-HCV positive liver cirrhosis. Clinical case

A case of hereditary hemochromatosis in a patient affected by anti-HCV positive liver cirrhosis is described. The difficulties for an exact diagnosis are underlined. Really, it can be particularly difficult to make a differential diagnosis between hereditary hemochromatosis and secondary hemochromatosis, if liver cirrhosis has already been found. Practically, at this stage of disease, the histological and clinical aspects of these two forms become completely interchangeable. Moreover, diagnostic difficulties increase when, at the same time, the patient presents more causes of potential liver damage. In this case report, the DNA-analysis, obtained by polymerase chain reaction amplification and enzymatic digestion, allows to make the diagnosis of hereditary hemochromatosis, because it showed the presence of two genetic mutations, considered responsible for the disease. Both the hereditary hemochromatosis and the HCV infection, had greatly contributed to the development of liver cirrhosis. In the future, DNA-analysis by amplification with polymerase chain reaction, can assume relevant importance for the screening of affected patients' first grade parents too. It could permit an early diagnosis of hereditary hemochromatosis and then to start a timelier and more efficacious therapy, to prevent an irreversible histological damage.     

Benefits of colonoscopic surveillance and prophylactic colectomy in patients with hereditary nonpolyposis colorectal cancer mutations

BACKGROUND: Predisposition genetic testing is now possible for many hereditary cancer syndromes, including hereditary nonpolyposis colorectal cancer. The optimal management of the elevated risk for cancer in carriers of mutations for hereditary nonpolyposis colorectal cancer is unclear. OBJECTIVE: To assess the life expectancy and quality-adjusted life expectancy benefits derived from endoscopic surveillance and prophylactic colectomy for persons who carry a mutation associated with hereditary nonpolyposis colorectal cancer. DESIGN: Decision analysis model. Lifetime risk for colorectal cancer, efficacy of surveillance and colectomy, stage-specific colorectal cancer mortality, and quality of life were included in the model. SETTING: Decision about a cancer prevention strategy at the time of a positive result on genetic testing. PATIENTS: Carriers of a mutation for hereditary nonpolyposis colorectal cancer who were 25 years of age. INTERVENTIONS: Immediate prophylactic colectomy; delayed colectomy on the basis of age, adenoma, or diagnosis of colorectal cancer; and endoscopic surveillance. Prophylactic surgical options were proctocolectomy with ileoanal anastomosis and subtotal colectomy with ileorectal anastomosis. MEASUREMENTS: Life expectancy and quality-adjusted life expectancy. RESULTS: All risk-reduction strategies led to large gains in life expectancy for carriers of a mutation for hereditary nonpolyposis colorectal cancer, with benefits ranging from 13.5 years for surveillance to 15.6 years for prophylactic proctocolectomy at 25 years of age compared with no intervention. The benefits of colectomy compared with surveillance decreased with increasing age and were minimal if colectomy was performed at the time of colorectal cancer diagnosis. When health-related quality of life was considered, surveillance led to the greatest quality-adjusted life expectancy benefit (3.1 years compared with proctocolectomy and 0.3 years compared with subtotal colectomy). CONCLUSIONS: Colonoscopic surveillance is an effective method of reducing risk for cancer in carriers of a mutation for hereditary nonpolyposis colorectal cancer. The individual patient's choice between prophylactic surgery and surveillance is a complex decision in which personal preferences weigh heavily.     

Nonsense mutations in the OCRL-1 gene in patients with the oculocerebrorenal syndrome of Lowe

A candidate gene, OCRL-1, for the oculocerebrorenal syndrome of Lowe (OCRL) has been identified via positional cloning strategies. We have now developed RT-PCR techniques which allow amplification of nearly all of the open reading frame from total RNA and have used the PCR products for mutational analysis. Single strand conformational polymorphism analysis detected aberrant migration in two unrelated patients, both of whom were shown to have the same nonsense mutation at base 2746 on direct sequencing. An additional patient was found to be missing a segment from his RNA that corresponds to an entire exon. The identification of mutations in the OCRL-1 gene provides strong genetic evidence for its being the gene involved in Lowe syndrome.     

A two-amino acid insertion in the Cys146- Cys167 loop of the alphaIIb subunit is associated with a variant of Glanzmann thrombasthenia. Critical role of Asp163 in ligand binding

The ligand binding site(s) of the alpha subunit of integrin alphaIIb beta3 (GPIIb-IIIa), a prototypic non-I domain integrin, remains elusive. In this study, we have characterized a Japanese variant of Glanzmann thrombasthenia, KO, whose platelets express normal amounts of alphaIIb beta3. KO platelets failed to bind the activation-independent ligand-mimetic mAb OP-G2 and did not bind fibrinogen or the activation-dependent ligand-mimetic mAb PAC-1 following activation of alphaIIb beta3 under any condition examined. Sequence analysis of PCR fragments derived from KO platelet mRNA revealed a 6-bp insertion leading to a 2-amino-acid insertion (Arg-Thr) between residues 160 and 161 of the alphaIIb subunit. Introduction of the insertion into wild-type recombinant alphaIIb beta3 expressed in 293 cells led to the normal expression of alphaIIb beta3 having the defect in ligand binding function. The insertion is located within the small loop (Cys146-Cys167) in the third NH2-terminal repeat of the alphaIIb subunit. Alanine substitution of each of the oxygenated residues within the loop (Thr150, Ser152, Glu157, Asp159, Ser161, and Asp163) did not significantly affect expression of alphaIIbbeta3, and only Asp163AlaalphaIIb beta3 abolished the ligand binding function. In addition, Asp163AlaalphaIIb beta3 as well as KO mutant alphaIIb beta3 constitutively expressed the PMI-1 epitope. Our present data suggest that Asp163 of the alphaIIb subunit is one of the critical residues for ligand binding.