Dissertations / Theses on the topic 'Haemochromatosis'

Haemochromatosis is the most common single gene disorder to afflict North- West European populations. It is probably the most common genetic disorder of iron metabolism worldwide. As many as 1 in 250 people in the UK are affected and although the phenotype causes only a mild increase in gastrointestinal iron absorption a proportion of affected individuals will accumulate sufficient iron over their life-time to cause cirrhosis and hepatocellular carcinoma. Venesection treatment instituted before cirrhosis has established ensures a normal life expectancy, but clinical presentation is often late in life after irreversible organ injury has occurred. Identification of people at risk in the early, asymptomatic stage by measurements of iron status is unreliable. The genetic defect responsible for haemochromatosis has been sought in the hope that its identification might facilitate early diagnosis and that studies on the gene product would lead to a greater understanding of the mechanisms of mammalian iron absorption. Genetic linkage to HLA-A3 placed the gene responsible for haemchromatosis in, or close to, the major histocompatibilty complex (MHC) on the short arm of chromosome 6 and a positional cloning strategy has been adopted. This thesis describes work directed to the identification of the haemochromatosis gene by positional cloning. The region telomeric to the MHC was mapped using yeast artificial chromosomes, from which new microsatellites were isolated. These markers were used in linkage disequilibrium analyses and the mapping of a recombination breakpoint that defined a haemochromatosis gene region. This region was physically mapped in fine detail and positional candidates sought by EST database analysis. Before a systematic search for genes in the region began a strong positional candidate was reported (Feder et al 1996). Analysis of this mutation in patients from the UK confirmed this to be the ancestral haemochromatosis mutation.

Hereditary haemochromatosis is characterized by unregulated intestinal iron absorption but the molecular mechanism by which this occurs is not understood. Most patients are homozygous for a disabling mutation, C282Y, in the HFE gene. Lately, mutations in a newly-identified second transferrin receptor gene (TfR2) have been associated with haemochromatosis. The physiological function of the HFE protein is unknown although it appears to mediate cellular transferrin-iron uptake in vitro. The divalent metal transporter (DMT1) is implicated in the mucosal uptake of ferrous iron but whether this process is regulated by HFE is not clear. To examine interactions between HFE and iron-transport proteins, rabbit and avian antibodies specific for HFE, DMT1 and TfR2 were generated. Confocal microscopy in human intestinal Caco-2 cells showed that endogenous HFE and DMT1 localise to separate vesicular compartments and that HFE interacts directly with both TfR1 and TfR2 in early endosomes. Within human duodenal mucosa TfR2 protein expression was restricted to the undifferentiated crypts where colocalisation with HFE occurred. The functional effects of HFE deficiency were investigated in an authentic genetic mouse model of hereditary haemochromatosis. The V_max for unidirectional ferrous iron uptake was greater in HFE-knockout mice than in matched wild-type controls (29.8 ± 4.6 compared with 13.0 ± 1.0 pmol/mg/min, p<0.01). Immunoreactive DMT1 protein was increased two-fold in duodenal mucosal extracts from HFE-knockout mice compared with wild-type, and pre-treatment of HFE-knockout mucosa with antibody to DMT1 abolished the increase in V_max. Uptake of iron presented as Fe³⁺ was enhanced, at low/physiological concentrations, in HFE-knockout mice (p<0.05) and studies using the Fe²⁺ -chelator ferrozine showed that mucosal ferric reductase activity reduced Fe³⁺ to Fe²⁺ for uptake.

Thesis (MSc)--University of Stellenbosch, 2008.
ENGLISH ABSTRACT: Iron metabolism disorders comprise the most common disorders in humans. Hereditary haemochromatosis (HH) is a common condition resulting from inappropriate iron absorption. The most common form of the disease (Type 1) is associated with mutations in the HFE gene. The C282Y homozygous genotype accounts for approximately 80% of all reported cases of HH within the Caucasian population. A second HFE mutation, H63D, is associated with less severe disease expression. The C282Y mutation is extremely rare in Asian and African populations. The H63D mutation is more prevalent and has been observed in almost all populations. Iron overload resulting from haemochromatosis is predicted to be rare in Asian Indian populations and is not associated with common HFE mutations that are responsible for HH in the Caucasian population. The aberrant genes associated with HH in India have not yet been identified. The present study attempted to identify variants in six iron regulatory genes that were resulting in the Type 1 HH phenotype observed in two Asian Indian probands from a highly consanguineous family. The promoter and coding regions of the HMOX1, HFE, HAMP, SLC40A1, CYBRD1 and HJV genes were subjected to mutation analysis. Gene fragments were amplified employing the polymerase chain reaction (PCR) and subsequently subjected to heteroduplex single-strand conformational polymorphism (HEX-SSCP) analysis. Samples displaying aberrations were then analysed using bi-directional semi-automated DNA sequencing analysis to identify any known or novel variants within the six genes. Variants disrupting restriction enzyme recognition sites were genotyped employing restriction fragment length polymorphism (RFLP) analysis. Mutation analysis of the six genes revealed 24 previously identified variants, five novel variants (HFE: 5’UTR-840T→G, CYBRD1: 5’UTR-1813C→T, 5’UTR-1452T→C, 5’UTR- 1272T→C; HJV: 5’UTR-534G→T, 5’UTR-530G→T), one previously described microsatellite and two novel repeats. Variants identified within the SLC40A1, CYBRD1 and HJV genes do not seem to be associated with the iron overload phenotype. A previously described HAMP variant (5’UTR-335G→T) was observed in the homozygous state in both probands. This variant seems to be the genetic aberration responsible for iron overload in this Indian family. The severe juvenile haemochromatosis phenotype usually associated with HAMP mutations, was not exhibited by the two Indian probands. Their symptoms resembled those observed in classic Type 1 HH. It is suggested that variants identified in the HMOX1 and HFE genes are modifying the effect of the HAMP variant and resulting in the less severe disease phenotype. Although this variant has only been identified in one Indian family, it could shed some light in the hunt for the iron-loading gene in India.
AFRIKAANSE OPSOMMING: Oorerflike hemochromatose (OH) is ‘n algemene siektetoestand wat ontstaan as gevolg van oneffektiewe opname van yster in die liggaam. Die mees algemene vorm van die siekte (Tipe 1) word geassosieer met mutasies in die HFE-geen. Die C282Y homosigotiese genotipe is verantwoordelik vir ongeveer 80% van alle gerapporteerde gevalle van OH binne die Kaukasiese bevolking. ‘n Tweede HFE mutasie, H63D, word geassosieer met minder ernstige siekte simptome. Die C282Y mutasie is besonder skaars in Asiese en Afrika bevolkings. Daar word bespiegel dat oorerflike ysteroorlading as gevolg van hemochromatose skaars is in Asiese Indiër bevolkings en word nie geassosieer met algemene HFE mutasies wat verantwoordelik is vir OH in Kaukasiese bevolkings nie. Die abnormale gene wat wél geassosieer word met OH in Indië is tot dusver nog nie identifiseer nie. Die doel van hierdie studie was om die variante in ses yster-regulerende gene te identifiseer wat die Tipe 1 OH fenotipe in hierdie familie veroorsaak. Hierdie fenotipe is waargeneem in twee Asies Indiese familielede afkomstig van ‘n bloedverwante familie. Die promotor en koderingsareas van die HMOX1, HFE, HAMP, SLC40A1, CYBRD1 en HJV gene is gesif vir mutasies. Geen fragmente is geamplifiseer met behulp van die polimerase kettingsreaksie (PKR) en daarna aan heterodupleks enkelstring konformasie polimorfisme (HEX-SSCP) analise blootgestel. PKR produkte wat variasies getoon het, is daarna geanaliseer deur tweerigting semi-geoutomatiseerde DNS volgorde-bepalingsanalise om enige bekende of nuwe variante binne die ses gene te identifiseer. Variante waar restriksie ensiem herkenningsetels teenwoordig is, is verder analiseer met behulp van die restriksie fragment lengte polimorfisme (RFLP) analise sisteem. Mutasie analise van die ses gene het 24 bekende variante, vyf nuwe variante (HFE: 5’UTR- 840T→G, CYBRD1: 5’UTR-1813C→T, 5’UTR-1452T→C, 5’UTR-1272T→C, HJV: 5’UTR-534G→T, 5’UTR-530G→T), een bekende herhaling en twee nuwe herhalings gewys. Variante wat binne die SLC4041, CYBRD1 en HJV gene geïdentifiseer is, blyk nie om by te dra tot die ysteroorladings-fenotipe nie. Die bekende HAMP variant (5’UTR-335G→T) is waargeneem in die homosigotiese toestand in beide van die aangetaste individue. Hierdie variant blyk om die genetiese fout te wees wat verantwoordelik is vir die ysteroorlading in die betrokke Indiese familie. Die erge juvenielehemochromatose fenotipe wat meestal geassosieer word met HAMP-mutasies, is nie waargeneem in hierdie familie nie. Hul simptome kom ooreen met die simptome van die klassieke Tipe 1 OH. Dit blyk moontlik te wees dat die variante identifiseer in die HMOX1 en HFE gene die impak van die HAMP variant modifiseer en die matiger siekte-fenotipe tot gevolg het. Alhoewel hierdie variant slegs in een Indiese familie geïdentifiseer is, kan dit lig werp op die soektog na die veroorsakende ysterladingsgeen in Indië.

Thesis (MSc)--Stellenbosch University, 2000.
ENGLISH ABSTRACT: Hereditary haemochromatosis (HH) is an autosomal recessive iron storage disease where the accumulation of iron in parenchymal organs may lead to diabetes, heart failure, liver cirrhosis, arthropathy, weakness and a variety of other ailments if preventive measures are not taken. HH is often not considered as a cause of these conditions, particularly not in the elderly where the background frequencies of type II diabetes, osteoarthritis and heart failure are generally high. Heterozygosity for C282Y, the HFE-mutation causing HH in approximately 80% of affected individuals worldwide, has been linked to a raised incidence of malignancies of the colon and rectum, stomach and the haematological system. One of the highest carrier-frequencies (116) in the world for this mutation has been reported in the South-African Afrikaner population, resulting in C282Y-homozygosity in approximately 1 in every 115 people in this group. A sample of 197 elderly Afrikaner volunteers was recruited for genotype/phenotype association studies. Their clinical presentation was denoted, biochemical iron-status determined and HFE genotyping performed. Either an increase or decrease in survival, or both, were proposed, depending on possible gender effects. HH has been positively associated with various cancer types, but may also protect against iron-deficiency anaemia which is by far the most frequent cause of anaemia in the older person. This study has led to the following findings: 1. The carrier frequency of mutation C282Y was found to be 1/8 in the elderly population (similar in males and females), which is slightly lower than the 1/6 reported in younger adults from the same population. Only one C282Y homozygote and two C282YIH63D compound heterozygotes were detected, all of them female. 2. The prevalence of diabetes, heart disease, arthropathy or a combination of these conditions did not differ significantly in C282Y heterozygotes and the mutationnegative group. 3. Among 24 C282Y heterozygotes only one individual with rectal carcmoma was detected compared with two cases with rectal- and seven with colonic malignancies in 153 mutation-negative individuals. The single female C282Y homozygote identified suffered from both rectal and colon carcinoma and died approximately 6 months ago as a consequence of her colon malignancy. 4. Serum ferritin appears to be a highly unreliable parameter of iron status, particularly in the elderly where a variety of factors that may influence the levels are often present in elderly individuals. This may be due to ageing alone or as a result of multiple comorbidities. 5. Serum ferritin levels were lower than expected in elderly subjects with mutation C282Y and compound heterozygotes with both C282Y and H63D, which may be related to a variable penetrance of the HFE gene mutations. It is possible that variation in other genes exist that confer protection against iron-loading by gene-gene interaction. The probability that environmental factors (e.g. a low iron diet) are more important in this respect cannot be excluded, although this is considered less likely in the light of the fact that the same trend was observed in all mutation-positive elderly individuals. It is therefore highly likely that C282Y -positive subjects with significant iron loading have died before reaching their seventies, particularly since none of the males included in this study were homozygous or compound heterozygous for the mutations analysed. In conclusion, possession of a mutant HFE gene does not appear to confer a survival advantage in old age, neither does it seem that mutation carriers with significant ironloading are overlooked by the medical fraternity. Further investigations are warranted to shed more light on the contributions of gene-gene and gene-environment interaction in the clinical manifestation of Hll, and how these processes can be manipulated to prevent the symptoms of this largely underdiagnosed disease.
AFRIKAANSE OPSOMMING: Oorerflike hemochromatose (OH) is 'n outosomaal resessiewe yster-oorladingssiekte waar akkumulasie van yster in parenkimale organe kan lei tot suikersiekte, hartversaking, lewer sirrose, artropatie, moegheid en 'n verskeidenheid van ander probleme indien voorkomende maatreëls nie getref word nie. OH word gewoonlik nie oorweeg as moontlike oorsaak vir hierdie toestande nie, veral nie in ouer mense nie waar die agtergrond-frekwensie van tipe II diabetes, osteoartritis en hartversaking in elk geval hoog is. Heterosigositeit vir die HFE mutasie C282Y, wat OH veroorsaak in ongeveer 80% van geaffekteerde gevalle wêreldwyd, is geassosieer met 'n verhoogde voorkoms van kanker van die kolon, rektum, maag en ook die hematologiese sisteem. Van die hoogste draer frekwensies ter wêreld vir hierdie mutasie (1/6) is gevind in die Afrikaner populasie van Suid-Afrika, wat daarop dui dat 1 uit elke 115 mense in die groep homosigoties vir die C282Y mutasie kan wees. Eenhonderd sewe-en-negentig bejaarde Afrikaner vrywilligers het aan die studie deelgeneem wat daarop gemik was om genotipe/fenotipe korrelasies uit te voer. Die kliniese beeld van elke individu is gedokumenteer, die yster status biochemies bepaal en HFE genotipering uitgevoer. Die a priori veronderstelling was dat oorlewing sou toeneem of afneem, of beide, afhangende van die geslag van die individu. Daar is voorheen 'n verband gevind tussen OH en die ontwikkeling van bogenoemde maligniteite, maar aan die ander kant kan dit moontlik ook beskerm teen anemie as gevolg van yster gebrek, wat juis die mees algemene oorsaak van anemie in die ouer persoon is. Hierdie studie het tot die volgende bevindings gelei: 1. Die draer frekwensie van mutasie C282Y was 1/8 in die bejaardes (dieselfde in mans en vrouens), wat effens laer is as die 1/6 wat gerappoteer is in jonger volwassenes. Slegs een C282Y homosigoot en twee C282YIH63D saamgestelde heterosigote is opgespoor, en al drie was vroulik. 2. Die voorkoms van suikersiekte, hartsiekte, gewrigspyne of 'n kombinasie van hierdie aandoenings het nie betekenisvol verskil tussen die C282Y heterosigote en die mutasienegatiewe groep nie. 3. Daar was slegs een persoon met rektum karsinoom in die groep van 24 bejaarde C282Y heterosigote, terwyl daar twee gevalle met rektum kanker en sewe gevalle met kolon kanker gevind is onder die 153 mutasie-negatiewe individue. Die enkele vroulike C282Y homosigoot wat opgespoor is het beide rektum- en kolonkanker gehad en is ongeveer 6 maande vóór voltooing van die tesis oorlede aan haar kolon karsinoom. 4. Dit wil voorkom asof serum ferritien veral in bejaardes 'n hoogs onbetroubare maatstaf is vir yster status, aangesien dit deur 'n verskeidenheid faktore beïnvloed word wat dikwels in bejaardes aanwesig is as gevolg van veroudering of veelvuldige komorbiditeite. 5. Die serum ferritien vlakke was laer as verwag in sowel die bejaarde C282Y-homosigoot as in die twee saamgestelde heterosigote met mutasies C282Y en H63D, wat moonlik die gevolg is van die wisselende graad van penetrasie van HFE mutasies. Dit is moontlik dat variasie in ander gene beskerming bied teen yster-oorlading deur middel van geen-geen interaksie. Die moontlikheid dat omgewingsfaktore (soos 'n lae-yster dieet) 'n belangrike rol speel in hierdie verband kan nie uitgesluit word nie, hoewel dit minder waarskynlik lyk te wees in die lig van die feit dat dieselfde neiging waargeneem is in alle mutasie-positiewe bejaardes. Die kans is dus redelik groot dat individue met die C282Y mutasie en betekenisvolle yster oorlading oorlede is voordat hulle die sewentiger jare kon bereik, veral omdat geeneen van die mans wat ingesluit is in die studie homosigoot of 'n saamgestelde heterosigoot was vir die mutasies wat geanaliseer is nie. Opsommend wil dit voorkom asof die teenwoordigheid van 'n mutante HFE geen nie 'n beter oorlewingskans bied op ouer leeftyd nie, en dit blyk ook dat mutasie draers met betekenisvolle ysteroorlading nie deur dokters misgekyk word nie. Verdere navorsing is nodig om meer lig te werp op die bydrae van geen-geen- en geen-omgewing interaksie in die kliniese manifestasie van OH, en ook hoe hierdie prosesse gemanipuleer kan word om die simptome van hierdie onder -gediagnoseerde siekte te voorkom.

[Truncated abstract. Please see the pdf format for the complete text.] The discovery of the C282Y mutation and its role in the development of hereditary haemochromatosis has allowed a greater understanding into the effects of iron overload and its involvement in other conditions such as diabetes and heart disease. It has also allowed the better classification of heterozygotes, who were previously only diagnosed through the use of family studies. There are however, areas of conflict between phenotyping and genotyping methods. My research involved examining the relationship between Haemochromatosis and certain diseases such as diabetes and heart disease; genotyping versus phenotyping discrepancies and the possible interaction of secondary mutations. In Chapter 3 a population study was undertaken with the aim of comparing genotyping versus phenotyping methods as well as increasing general practitioner awareness regarding hereditary haemochromatosis and its diagnosis. It was determined that a minimum of 5000 subjects would be required to give the study sufficient power. Individuals were to be between the ages of 20—40 years, and thus presumably presymptomatic. Participation was entirely voluntary and a consent form was to be signed. Recruitment of subjects proved to be difficult and there was a selective bias towards individuals already displaying symptoms of haemochromatosis. In total less than a 100 subjects were recruited for the study. There were several issues encountered in the implementation of this study. Firstly the number of GPs participating was probably insufficient to recruit the subjects required. A more extensive campaign was probably required to enroll more GPs. Secondly it is very difficult for a busy GP to find the time necessary to explain the study to each of his patients and to get them to sign the consent form. Finally a bias developed in some of the requests. The subjects participating in this study were supposed to be random but in many cases the GPs had enrolled them in the study because they had symptoms of iron overload. In effect the biggest obstacle this study faced was the recruitment of subjects. Due to the small number of subjects little statistical data could be obtained from this study. It was noted, however, that genotyping methods detected two individuals who were homozygous for the C282Y mutation. Both also had increased transferrin saturation levels. Phenotyping detected 5 individuals with increased transferrin saturation. The three others detected via phenotyping were C282Y heterozygotes. Haemochromatosis has long been though to be related to the development of diabetes due to the effect of iron overload on the pancreas. If this is so it would be logical to assume that the prevalence of haemochromatosis would be higher in a diabetic population. Chapter 4 examined the possibility that diabetics have a higher frequency of the C282Y mutation. A population group consisting of 1355 diabetics was genotyped for the C282Y mutation and iron studies were performed on all heterozygotes and C282Y homozygotes. Initial findings indicated that there was a significant difference between the diabetic and control population. However, this finding was the opposite of what was expected, there seemed to be a decreased frequency of the Y allele in the diabetic population rather than an increased one. The control and diabetic populations were not matched in terms of ethnicity. The removal of the ethnic bias in the diabetic population altered the statistics so there was no longer a significant difference between the two groups. This study highlighted the importance of using appropriate control populations as comparison groups. The final results of the study indicated that there was no significant difference between the diabetic population and the control population. This would seem to indicate that there is not an increased occurrence of the C282Y mutation in the diabetic population when compared to the control group. Chapter 5 considered the possible association between C282Y heterozygosity and cardiovascular disease as well as the potential for early mortality. Several recent studies have indicated that C282Y heterozygosity may be a risk factor for the development of atherosclerosis, possibly on the basis of increased iron loading. Using a control population and a population of individuals with known coronary events the incidence of the C282Y mutation was compared against other risk factors. C282Y heterozygosity did not appear to be a risk factor for atherosclerosis. There was however, a statistically significant link between increased ferritin in women and carotid plaques. A population of elderly women was genotyped in order to examine the effects of C282Y heterozygosity on longevity. The first hypothesis addressed in chapter 5 was that C282Y heterozygosity was a risk factor for the development of coronary heart disease.

Hereditary haemochromatosis is an autosomal recessive disorder of iron metabolism, characterised by increased iron absorption and progressive iron accumulation particularly in the liver. It has been shown that hepatocytes can acquire iron in two forms; transferrin-bound iron (TBI) and non-transferrin-bound iron (NTBI)1. Known transporters of NTBI into the cell include DMT1 and ZIP14, and FPN is the only transporter known to export iron. The aims of this study were to characterize the zinc transporter, ZnT-1 and determine whether it is involved in iron transport, specifically as an exporter. There was a decrease in mRNA expression of the short untranslated isoform of ZnT-1 in double mutant (Hfe -/- and TfR2 Y245X) mouse liver, a trend also seen in TfR1 and Hamp. The long untranslated isoform, however, was significantly higher in the iron-deficient mice as was expression of TfR1 and Ferroportin. Iron and zinc efflux was measured in cells over-expressing ZnT-1 and control cells. There was no difference between over-expressed and control cells in iron efflux. However, at 60 min, over-expressed cells had significantly more zinc efflux than control cells. More zinc than iron was released from the cell. The results of this study do not support the hypotheses that (i) ZnT-1 reduces intracellular cytoplasmic iron concentration by promoting efflux and 2 ZnT-1 is down-regulated in iron-loaded cells.

This dissertation focused on identifying novel chemical and genetic modulators of iron homeostasis. Iron is an essential element for human health. Disorders such as anaemia and haemochromatosis can develop when iron levels are not maintained within a normal physiological range. The findings of this program included the identification of a new iron chelating compound, demonstration of iron chelation in a haemochromatosis mouse model by a flavonol, identification of iron metabolism-related genes and variants which may assist in distinguishing suitable blood donors, and the identification of novel genes which may contribute to modulating iron homeostasis by regulating the iron exporter ferroportin.

Iron is essential for virtually all organisms, yet it can be highly toxic if not properly regulated. Only the Lyme disease pathogen Borrelia burgdorferi has evolved to not require iron (Aguirre et al., 2013).Recent findings have characterised elements of the iron metabolism network, but understanding of systemic iron regulation remains poor. To improve understanding and provide a tool for in silico experimentation, a computational model of human iron metabolism has been constructed. COPASI was utilised to construct a model that included detailed modelling of iron metabolism in liver and intestinal cells. Inter-cellular interactions and dietary iron absorption were included to create a systemic computational model. Parameterisation was performed using a wide variety of literature data. Validation of the model was performed using published experimental and clinical findings, and the model was found to recreate quantitatively and accurately many results. Analysis of sensitivities in the model showed that, despite enterocytes being the only route of iron uptake, almost all control over the system is provided by reactions in the liver. Metabolic control analysis identified key regulatory factors and potential therapeutic targets. A virtual haemochromatosis patient was created and compared to a simulation of a healthy human. The redistribution of control in haemochromatosis was analysed in order to improve our understanding of the condition and identify promising therapeutic targets. Cellular prion protein (PrP) is an enigmatic protein, implicated in disease when misfolded, but its physiological role remains a mystery. PrP was recently found to have ferric-reductase capacity. Potential sites of ferric reduction were simulated and the findings compared to PrP knockout mice experiments. I propose that the physiological role of PrP is in the chemical reduction of endocytosed ferric iron to its ferrous form following transferrin receptor-mediated uptake.

Iron overload cardiomyopathy (IOC) has been recently described as a dilated cardiomyopathy, characterized by left ventricular (LV) remodelling with chamber dilatation and reduced LV ejection fraction (LVEF). However, primary haemochromatosis, a genetically determined condition leading to iron overload, is classically categorized as an infiltrative cause of cardiomyopathy. Moreover, secondary haemochromatosis may lead to severe diastolic LV dysfunction in the early stages of the disease, before LVEF is affected. In this study, we describe the forms, pathophysiology, and genotypic expressions of HFE and hepcidin (HAMP) gene mutations focusing on the possibility of digenic occurrence that could lead to potential development of iron overload cardiomyopathy among Irish patients and their direct family members. The prevalence of iron overload cardiomyopathy (IOC) in Irish population is increasing. The spectrum of symptoms of IOC varied. Early in the disease process, patients may be asymptomatic, whereas severely overloaded patients can have terminal heart failure complaints that are refractory to treatment. It has been shown that early recognition and intervention may alter outcomes. In this study the combination of cardiac biomarkers (troponin I and creatine kinase), iron studies (serum ferritin, serum hepcidin, and transferin saturation) level determinations and genotyping of the HFE (C282Y/H63D) and hepcidin HAMP (C70R) gene mutations were carried out and allele frequencies were correlated within the Irish population. The first finding of this study was a trend towards significant elevation of iron studies: serum ferritin level, percent transferin saturation and decreasing serum hepcidin observed in dilated cardiomyopathy patients and direct family members identified with HFE C282Y/H63D and HAMP C70R heterozygotes who were asymptomatic. This was not associated with age and suggests that there is a threshold level of iron studies above which symptoms occur. This discordance between the symptomatic and asymptomatic HFE C282Y/H63D and HAMP C70R heterozygous formed the basis of subsequent analyses. The second finding of this study was a trend towards significant elevation of the selected cardiac biochemical markers from the median level of serum troponin-I and creatine kinase observed in patients and direct family members with HFE C282Y/H63D and HAMP C70R heterozygous who were again asymptomatic. This was not associated with age and cardiac complaints or history and suggests that there is a threshold level of cardiac biochemical marker activities that triggers the condition and as predisposing factors to a potential development of iron overload cardiomyopathy. The genotypic expressions of HFE and HAMP were identified and showed that among the Irish patients diagnosed with dilated cardiomyopathy and their family members, a significant trend of digenic occurrence of both mutations. The heterozygous C282Y/H63D and C70R revealed in this study that it is a predisposing factors developed at certain age of life.

Dans la cavité buccale, la santé parodontale repose sur un équilibre entre la communauté bactérienne sous-gingivale et la fonction immunitaire de l’hôte, toutes deux compatibles avec un état sain. Le modèle pathophysiologique actuel de la parodontite chronique montre un cercle vicieux entre un microbiote dysbiotique et une dérégulation de la réponse inflammatoire. Alors qu’un nombre croissant d’associations entre maladie parodontale et pathologie systémique sont rapportées, ce travail décrit comment une maladie génétique de surcharge en fer — l’hémochromatose héréditaire liée au gène HFE — est susceptible d’influencer l’équilibre hôte–microbiote au sein du sillon sous-gingival. Les études transversales, clinique et microbiologique, que nous avons menées, ont ainsi montré un impact du coefficient de saturation de la transferrine sur la sévérité de la parodontite, probablement en lien avec la sélection d’espèces pathogènes pour le parodonte. Cependant, l’analyse morphométrique du modèle murin de l’hémochromatose héréditaire Hfe(–/–) a révélé des altérations de la micro-architecture de l’os alvéolaire mandibulaire, suggérant un dérèglement du remodelage osseux. Un croisement entre deux modèles animaux, parodontite induite par Porphyromonas gingivalis et Hfe(–/–), a donc été mis en œuvre, afin d’explorer les mécanismes de cette association entre excès de fer, dysbiose bactérienne et parodontite
In the oral cavity, periodontal health relies on a balance between the subgingival bacterial community and the host’s immune function. The current pathophysiological model of chronic periodontitis shows a negative feedback loop that implies a dysbiotic microbiota and a dysregulation of the inflammatory response. As an increasing number of associations between the periodontal disease and systemic conditions are reported, this work describes how a genetic iron overload disease — HFE-related hereditary haemochromatosis — is likely to influence the host–microbiota interactions within the subgingival sulcus. We conducted clinical and microbiological cross-sectional studies, that showed transferrin saturation coefficient having an impact on the severity of periodontitis, probably related to the promotion of periodontal pathogenic species. However, morphometric analysis of the murine model for hereditary haemochromatosis Hfe(–/–) revealed alterations in the mandibular alveolar bone micro-architecture, suggesting a disruption of bone remodeling. A mixed animal model, with Porphyromonas gingivalis-induced periodontitis and Hfe(–/–) mice, was therefore performed, in order to explore the underlying mechanisms of the relationships between iron excess, bacterial dysbiosis, and periodontitis

This project was conducted to determine whether the genetic variance of an iron regulatory gene was associated with the development or severity of chronic venous leg ulceration. In addition, the study examined the association of wound exudate protein composition with both the genetic variance of the target gene and wound healing outcomes. The evidence collected supported the hypothesis that wound healing may be affected by dysregulation of iron. By virtue of the discovery phase protein profiling methods used, a wound fluid protein reference library was created, against which future studies may be searched to identify potential therapeutic targets.

Hereditary haemochromatosis (OMIM #235200) is a late onset disorder resulting from excess iron absorption. It causes symptoms such as bronze pigmentation of the skin, joint pain, abdominal pain, weight loss, impotence, cardiomyopathy, diabetes and may lead to cirrhosis of the liver. The most common cause of this disease in Caucasians is homozygosity for the C282Y mutation in the HFE gene on chromosome 6p21.3. The penetrance of the C282Y mutation is not complete with many environmental and genetic factors influencing the phenotypic expression of the disease. A number of SNPs in genes in the iron metabolic pathway previously associated with altered serum iron indices were tested in the current study. The results suggested that two of the SNPs, namely rs1799852 in Transferrin and rs884409 in CYBRD1 may contribute to altered SF levels in HFE-associated haemochromatosis. The first major finding of this study was a significantly higher serum ferritin (SF) level observed in iron-loaded male C282Y homozygotes who were symptomatic compared to those that were asymptomatic. This was not associated with age and suggests there is a threshold level of SF above which symptoms occur. This discordance between the symptomatic and asymptomatic C282Y homozygotes formed the basis of subsequent analyses. A second major finding was the association of the TNFalpha 21112 promoter haplotype with increased SF levels in symptomatic male C282Y homozygotes. To explore the mechanism underlying this association, luciferase assays were performed in the presence and absence of extracellular iron. The results revealed there was no definitive change in TNFalpha expression associated with the 21112 haplotype compared to the other haplotypes. The expression levels of a number of genes in the iron pathway (HFE, HAMP, TFRC, TFR2 and TNFalpha) were measured in lymphoblastoid cell lines derived from males of different TNFalpha haplotypes and HFE genotypes. The C282Y homozygotes analysed in this study included both symptomatic and asymptomatic samples. The major finding of the expression studies was that the TNFalpha 21112 haplotype was associated with altered TFRC expression levels specifically in the symptomatic C282Y homozygotes. This provides an explanation of the increased SF levels seen in these patients. While a mechanism for the modulation of TFRC expression by the TNFalpha haplotype remains unclear, this study has confirmed TNFalpha as a significant modifier gene in HFE-associated haemochromatosis. The discovery of modifier genes in both conventional and unconventional pathways of iron metabolism may allow for the development of novel preventative and treatment strategies in a new era for hereditary haemochromatosis.

Research Doctorate - Doctor of Philosophy (PhD)
Iron is essential for life, having critical roles in oxygen transport, cellular energy production and as an enzyme cofactor, however too much iron can be detrimental to health. Approximately 10% of people in developed countries have body iron levels above normal reference ranges. This generally arises as a result of excessive dietary iron intake or genetic mutations that perturb systemic iron homeostasis, most commonly loss-of-function polymorphisms in the HFE gene. Abnormally high body iron levels can lead to the disorder haemochromatosis, which involves damage to the liver and possibly also other organs such as the heart and pancreas, however effects on the brain are not well understood. Although iron accumulation in particular brain regions due to certain rare genetic mutations is implicated in severe neurodegeneration and neurologic dysfunction, the blood-brain barrier is thought to protect the brain against the effects of high systemic iron levels in haemochromatosis. To investigate the effects of iron overload disorders on the brain, microarray technology was used to assess genome-wide brain gene expression in mouse models of dietary or genetic iron overload. Three groups of mice were studied: wildtype control mice, wildtype mice fed a short-term iron-supplemented diet and mice with disruption of the Hfe gene (Hfe knockout). As there are many methods available for analysing microarray data, a range of different approaches were first evaluated for use with these datasets. To improve the robustness of the findings, several of the most suitable approaches were utilised for subsequent analyses. Various bioinformatics tools were then used to determine potential functional effects of the observed changes in gene expression. Select expression changes of interest were validated by real-time reverse transcription polymerase chain reaction (RT-PCR). Neither model of iron overload showed increased brain non-haem iron levels nor expression changes for a large number of iron-related genes, however both models did show potentially important alterations in brain gene expression. Mice fed a short-term high-iron diet showed only restricted changes relative to control mice, however there was altered expression of genes relating to biological functions involving iron, such as nitric oxide signalling and accumulation of the lysosomal waste product lipofuscin. Lipofuscin production is accelerated by high iron levels and excessive lipofuscin is associated with neurodegeneration in the disease class neuronal ceroid lipofuscinoses. Iron-supplemented mice also showed expression changes for a number of genes causatively linked to other rare neurologic disorders. Similarly, the Hfe knockout mouse model of genetic haemochromatosis showed brain gene expression changes relating to lysosomal lipofuscin accumulation and certain neurologic disorders. However, in contrast to the dietary iron-supplemented mice, Hfe knockout mice showed extensive changes in brain gene expression relative to wildtype controls. These included expression changes for genes involved in key brain functions, such as neurotransmission, synaptic plasticity and transcriptional regulation, as well as processes that are influenced by iron, such as haem synthesis and degradation. In addition, analysis of molecular pathways revealed a disproportionately high number of expression changes for genes relating to Alzheimer’s disease, suggesting disruption of the Hfe gene might influence disease processes. There was also evidence for effects on related pathways such as Notch signalling, which could potentially impair memory and other cognitive functions independent of Alzheimer’s-related effects. Assessment of heart gene expression changes in these mouse models revealed few similarities with the brain, suggesting that many of the expression changes observed in the brain may be tissue-specific. However there were some notable changes in the heart that could have functional consequences, including expression changes for genes involved in cardiac pacemaking, intracellular calcium release and neurotransmitter degradation. Physiological investigations of sinoatrial node preparations from Hfe knockout mice were indicative of a lower heart rate at baseline compared to wildtype control mice but no difference in contractile responses to either stimulatory (noradrenaline) or inhibitory (carbachol) agents. Together these findings provide evidence for important brain and heart gene expression changes in disorders of iron overload. The nature and extent of these changes appears dependent on the cause (dietary or genetic) and duration (acute or chronic) of iron loading. These changes could have consequences for both normal functioning and disease pathogenesis and might help explain some of the problems experienced by patients with iron overload disorders. The findings suggest a range of new research directions and are likely to alter the way that haemochromatosis and other iron overload disorders are perceived by clinicians, possibly leading to improved monitoring and treatment of the large number of patients with these conditions.

A dissertation submitted to the Faculty of Medicine, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the Degree Master of Science in Medicine. Johannesburg, 1995
HLA-linked haemochromatosis is the result of an inborn error of metabolism inherited as an autosomal recessive gene, closely linked to the HLA locus on chromosome six. In this condition iron absorption is inappropriately high leading to iron overload. Integral to the pathogenesis of this disorder and in contrast to other causes of iron overload, is the relatively modest accumulation of iron within cells of both the small intestine and the reticuloendothelial system and the excessive deposition of iron in parenchymal cells of the liver and other organs. This observation has led to the suggestion that the primary defect(s) could be present in either the gut, the liver, the reticuloendothelial system or all three. Abnormalities in iron uptake by cells, iron transport through and between cells and iron storage in cells have all been suggested as possible mechanisms responsible for the abnormal absorption and distribution of iron in haemochromatosis. Malfunction of the iron transport protein transferrin or its receptor could be responsible for abnormal distribution and iron loading while an abnormality of ferritin iron storage could explain why some cells appear to be unable to store iron and others are iron overloaded.
IT2018