Made available in DSpace on 2014-12-17T14:16:20Z (GMT). No. of bitstreams: 1 GiocondaDRL.pdf: 1031402 bytes, checksum: 0016e69e527bddffea93909fa2748a03 (MD5) Previous issue date: 2007-08-29 / Hereditary Hemochromatosis (HH) is a genetic disease caused by high iron absorption and deposition in several organs. This accumulation results in clinical disturbances such as cirrhosis, arthritis, cardiopathies, diabetes, sexual disorders and skin darkening. The H63D and C282Y mutations are well defined in the hemochromatosis etiology. The aim of this paper was that of identifying the H63D and C282Y genetical mutations in the hemochromatosis gene and the frequency assessment of these mutations in the HFE protein gene in patients with hyperferritin which are sent to the DNA Center laboratory in Natal, state of Rio Grande do Norte. This paper also evaluates the HH H63D and C282Y gene mutations genotype correlation with the serum ferritin concentration, glucose, alanine aminotransferasis, aspartato aminotransferasis, gama glutamil transferasis and with the clinical complications and also the interrelation with life habits including alcoholism and iron overload. The biochemical dosages and molecule analyses are done respectively by the enzymatic method and PCR with enzymatic restriction. Out of the 183 patients investigated, 51,4% showed no mutation and 48,6% showed some type of mutation: 5,0% were C282Y heterozygous mutation; 1,1%, C282Y homozygous mutation; 31%, H63D heterozygous mutation; 8,7%, H63D homozygous mutation; and 3,3%, heterozygous for the mutation in both genes. As to gender, we observed a greater percentage of cases with molecular alteration in men in relation to women in the two evaluated mutations. The individuals with negative results showed clinical and lab signs which indicate hemochromatosis that other genes could be involved in the iron metabolism. Due to the high prevalence of hemochromatosis and taking into account that hemochromatosis is considered a public health matter, its gravity being preventable and the loss treatment toxicity, the early genetic diagnosis is indicated, especially in patients with high ferritin, and this way it avoids serious clinical manifestations and increases patients' life expectation. Our findings show the importance of doing such genetic studies in individuals suspected of hereditary hemochromatosis due to the high incidence of such a hereditary disease in our region / A hemocromatose heredit?ria (HH) ? uma doen?a gen?tica causada pela absor??o e deposi??o elevada de ferro em v?rios ?rg?os. Este ac?mulo resulta em complica??es cl?nicas como cirrose, artrite, cardiopatias, diabetes, desordens sexuais e escurecimento da pele. As muta??es H63D e C282Y est?o bem definidas na etiologia da hemocromatose. O objetivo deste trabalho foi a identifica??o das muta??es gen?ticas H63D e C282Y no gene da Hemocromatose e avalia??o da freq??ncia dessas muta??es no gene da prote?na HFE em pacientes com hiperferritinemia que s?o encaminhados ao laborat?rio DNA Center Natal / RN. Al?m disso, avaliar a correla??o dos gen?tipos das muta??es H63D e C282Y do gene da HH com a concentra??o s?rica da ferritina, glicose, alanina aminotransferase, aspartato minotransferase, gt e com as complica??es cl?nicas e ainda a interrela??o com os h?bitos de vida incluindo o etilismo e dieta com sobrecarga de ferro. As dosagens bioqu?micas e an?lises moleculares foram realizadas respectivamente atrav?s do m?todo enzim?tico e PCR com restri??o enzim?tica. Dos 183 pacientes investigados 51,4% apresentaram aus?ncia de muta??o e 48,6% com algum tipo de muta??o: 5,0% C282Y heterozigoto mutado; 1,1% C282Y homozigoto mutado; 31% H63D heterozigoto mutado; 8,7% H63D homozigoto mutado; e 3,3% heterozigoto para a muta??o em ambos os genes. Com rela??o ao sexo, observou-se o maior percentual de casos com altera??o molecular em homens em rela??o a mulheres nas duas muta??es avaliadas. Os indiv?duos com resultados negativos apresentaram sinais cl?nicos e laboratoriais indicativos de hemocromatose sugerindo que outros genes poder?o estar envolvidos no metabolismo do ferro. Devido ? alta preval?ncia da hemocromatose, e tendo em vista que a hemocromatose ? considerada um problema de sa?de p?blica, sua gravidade ser preven?vel e a baixa toxicidade do tratamento, o diagn?stico gen?tico precoce torna-se indicado, principalmente nos pacientes com ferritina elevada, e com isso evitar manifesta??es cl?nicas graves e aumentar a expectativa de vida dos pacientes com esta doen?a. Nossos achados mostram a import?ncia da realiza??o de estudos gen?ticos em indiv?duos com suspeita de hemocromatose heredit?ria em virtude de elevada incid?ncia dessa doen?a de cunho heredit?rio em nossa regi?o

L’hémochromatose HFE est une maladie du métabolisme du fer liée au gène HFE dont la principale mutation est C282Y. L’objectif général de ce travail était d’étudier l’influence de facteurs génétiques et non génétiques sur l’expression phénotypique de patients atteints d’hémochromatose HFE. Cette étude prospective incluait les patients C282Y/C282Y etC282Y/H63D inclus en protocole de saignées entre janvier 2004 et décembre 2011 au centre de santé brestois de l’EFS-Bretagne. Dans un premier temps, nous avons étudié l’influence du génotype C282Y/H63D sur la survenue d’une surcharge en fer. Nous avons confirmé que le variant H63D doit être considéré comme un facteur de susceptibilité dont l’expression est liée à la présence de co-facteurs responsables d’une hyper ferritinémie. Ensuite, nous avons étudié le rôle des grossesses et de l’alimentation sur l’expression phénotypique du génotype C282Yhomozygote. Nous avons montré qu’il existe bien une différence d’expressivité clinique liée au sexe chez les patients C282Y/C282Y. Cependant, nos données n’ont pas confirmé l’effet protecteur typiquement attribué aux grossesses pour expliquer la plus lente accumulation de fer chez les femmes. Cette étude a également mis en évidence une association modérée entre la consommation d’aliments riches en fer et le degré de surcharge en fer des patients C282Yhomozygotes traités par phlébotomies. Ce travail contribue à mieux comprendre l’hétérogénéité phénotypique observée dans l’hémochromatose HFE. La finalité est de pouvoir repérer précocement les sujets les plus à risque de développer les surcharges en fer les plus sévères et par conséquent des complications cliniques. / HFE hemochromatosis is a disorder of iron metabolism related to the HFE gene whose mainmutation is C282Y. The overall aim of this study was to investigate the influence of genetic and non genetic factors on phenotypic expression of patients with HFE hemochromatosis. This prospective study included the C282Y/C282Y and C282Y/H63D patients enrolled in a phlebotomy program between 2004 and 2011 in a blood centre of western Brittany (Brest, France). First, weassessed the weight of the C282Y/H63D genotype in the occurrence of iron overload. We confirmed that H63D is a discrete genetic susceptibility factor whose expression is most visible in association with other co-factors responsible for hyper ferritinemia. Then we investigated the effect of pregnancies and iron-rich diet on phenotypic expressivity of the C282Y/C282Y genotype. We have shown that there is a difference in clinical expression related to gender in C282Y/C282Ypatients. However our findings did not confirm that pregnancies protect against iron accumulationin women. This study established a moderate link between dietary iron intake and the degree of iron overload in HFE hemochromatosis patients who come to medical attention. This work contributes to a better understanding of the phenotypic heterogeneity observed in HFE hemochromatosis. The purpose is to identify precociously subjects the most at risk of developing iron overload and therefore clinical complications.

La Hemocromatosis Hereditaria (HH) es una enfermedad genética, autosómica recesiva, caracterizada por una absorción masiva de hierro procedente de la dieta. Con el tiempo el exceso de hierro se acumula en diferentes órganos como en hígado, páncreas, corazón, glándulas endocrinas y articulaciones produciendo la alteración de los mismos. La clínica característica de la Hemocromatosis Hereditaria se presenta con cirrosis hepática, que puede desembocar en un carcinoma hepatocelular, diabetes, arritmias, pigmentación oscura de la piel, atralgias, hipogonadismo e hipotiroidismo. La enfermedad se detecta mediante análisis bioquímicos de ferritina sérica y saturación de transferrina, una biopsia hepática y/o un estudio genético de las mutaciones del gen HFE, principalmente las mutaciones C282Y y H63D. La Hemocromatosis Hereditaria es una enfermedad que presenta un tratamiento sencillo y barato, que consiste en extracciones periódicas de sangre o flebotomías para reducir de esta manera el exceso de hierro que se ha acumulado dentro del organismo. Si el tratamiento se realiza antes de que se produzcan daños en los órganos internos se previene las complicaciones clínicas de la enfermedad y se reestablece la esperanza de vida del paciente, por lo que un diagnóstico precoz es esencial para llevar a cabo un tratamiento con éxito en esta enfermedad.En esta tesis se centra en la Hemocromatosis Hereditaria a nivel clínico y básico, y en ella se han abordado los siguientes puntos:- Detección de la frecuencia de las mutaciones C282Y y H63D del gen HFE en 107 pacientes con Hemocromatosis Hereditaria y en controles de la población española.- Se describe que el genotipo C282Y/H63D es más frecuente en pacientes con Hemocromatosis Hereditaria que en controles y que la mayoría de los pacientes con Hemocromatosis Hereditaria (91,6%) presentan los genotipos C282Y/C282Y o C282Y/H63D del gen HFE.- Cribado de 5370 donantes de sangre para las mutaciones C282Y y H63D del gen HFE, en el que se detectan 8 individuos C282Y/C282Y y 74 individuos C282Y/H63D. Se describe la penetrancia de los genotipos C282Y/C282Y y C282Y/H63D.- Detección de parámetros bioquímicos de saturación de transferrina, hierro sérico y ferritina sérica en grupos de individuos con genotipos concretos, detectándose diferencias significativas entre el genotipado C282Y/C282Y y los otros genotipos. - Búsqueda de nuevas mutaciones en 9 pacientes con Hemocromatosis Hereditaria sin los genotipos C282Y/C282Y ni C282Y/H63D en los genes HFE, TFR2 y FPN1. Se describe la existencia de diversos polimorfismos.- Secuenciación de la región promotora del gen HFE en 25 pacientes Italianos de Hemocromatosis Hereditaria sin las mutaciones C282Y ni H63D. Se detecta el cambio -48 C/G en el 5'UTR del gen HFE en un paciente italiano de Hemocromatosis Hereditaria sin las mutaciones C282Y ni H63D.- Estudio comparativo de la zona promotora del gen HFE en humanos, rata y ratón, detectándose elementos reguladores conservados mediante técnicas de comparación computacionales. Identificación de 1398 pb correspondientes a la región promotora del gen HFE de rata y detección de regiones reguladoras del promotor de rata mediante experimentos de retardación en gel. Se estudia el promotor humano del gen HFE mediante experimentos de "Reporter gene" con luciferasa.- Detección de 5 formas de "splicing" en el gen HFE humano mediante experimentos de RT-PCR en la línea celular HepG2.- Se alarga la región 3' UTR del gen HFE humano (exón 7) en 1512 pares de bases y la región 3' UTR del gen HFE de ratón (exón 6) en 1990 pares de bases mediante experimentos de RT-PCR, 3' RACE y "Northern Blot".- Detección de 2 nuevas formas de terminación del gen HFE humano en el exón 6 mediante experimentos de 3' RACE.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2016-01-04T19:56:38Z No. of bitstreams: 1 GiocondaDiasRodriguesLeao_TESE.pdf: 44025376 bytes, checksum: 9da025324f9108141027f1f3a1cc3e16 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2016-01-05T20:29:30Z (GMT) No. of bitstreams: 1 GiocondaDiasRodriguesLeao_TESE.pdf: 44025376 bytes, checksum: 9da025324f9108141027f1f3a1cc3e16 (MD5) / Made available in DSpace on 2016-01-05T20:29:30Z (GMT). No. of bitstreams: 1 GiocondaDiasRodriguesLeao_TESE.pdf: 44025376 bytes, checksum: 9da025324f9108141027f1f3a1cc3e16 (MD5) Previous issue date: 2013-05-17 / Background & Aims: HFE-associated Hereditary Hemochromatosis (HH) is one of the most frequent autosomal recessive disease in the caucasian population, caused by the high absorption and deposition of iron in several organs. This accumulation results in several clinical complications such as cirrhosis, arthritis, cardiopathies, diabetes, sexual disorders and skin darkening. Although most of the cases are homozygous individuals for the C282Y mutation, another two mutations, H63D and S65C, have been reported to be associated with milder forms of the disease. The objective is to avaluate the distribution of C282Y, H63D and S65C mutations in the HFE gene in patients with suspected HH in the state of Rio Grande do Norte, Brazil. Methods: Samples of peripheral blood were taken from 335 patients originating from Natal-RN, a city in northeastern Brazil with suspected of HH and which were screened for the HFE gene C282Y, H63D and S65C mutations, using molecular genetics assays (Polymerase Chain Reaction- Restriction Fragments Length Polymorphism). The main criterion for including such patients in the study was the increasing of persistent serum ferritin in individuals aged between 18 and 70 or older, both males and females. As to the exclusion criteria, individuals holding hemolytical anemia, talassemy and previously report of blood transfusion did not take part of the study. Results: Out of the 335 patients studied, 143 patients showed absence of mutation and 195 showed some kind of mutation in the HFE gene: 07/335 (2,08%) were homozigous C282Y, 25/335 heterozygous C282Y, 25/335 (7,46%) were homozigous H63D, 115/335 (34,32%) heterozygous H63D, 5/335 (1,48%) heterozygous S65D, 11/ 335 (3,28%) and were double heterozygous (H63D/C282Y). None patients were Homozygous S65D and S65D heterozygous (S65D/H63D and S65D/C282Y). Conclusions. The distribution of the HFE gene C282Y, H63D and S65C mutations found in our group matches the tendencies observed in other European countries. Due to the high prevalence of hemochromatosis, its seriousness and easy treatment, the genetic diagnosis of HH has become a dream, especially in the high risk group.

Le cancer épithélial de l’ovaire est le cancer gynécologique le plus agressif avec le plus haut taux de mortalité. La croissance des cellules cancéreuses de l’ovaire est limitée par les nutriments de l’environnement, le fer étant un des éléments indispensables à leur prolifération. L’hémochromatose héréditaire est une maladie associée à une accumulation corporelle de fer. Cette maladie est liée à deux mutations majeures du gène HFE soit H63D et C282Y. Étant donnée l’influence de la protéine HFE sur l’entrée du fer dans la cellule, des mutations du gène HFE pourraient être associées à une croissance rapide des cellules cancéreuses. Des études de génotypage du gène HFE effectuées chez 526 patientes avec cancer épithélial de l’ovaire, ont révélées une fréquence allélique de la mutation C282Y significativement plus élevées chez les patientes avec tumeur ovarienne comparativement aux patientes du groupe contrôle (5.9% versus 1.3%, p = 0.02). De plus, le taux de survie des patientes avec mutations C282Y et tumeur ovarienne de G3, après 2 ans, est faible (20%) lorsque comparé à celui des patientes sans mutations (60%, p = 0.005). Une analyse de régression multivariée de Cox a démontrée un risque relatif de 3.1, suggérant que les patientes avec mutations C282Y ont 3 fois plus de chance d’avoir une faible survie (p=0.001). Également, des études de corrélation ont démontrées que les niveaux de ferritine du sérum étaient plus élevés chez les patientes avec grade avancé du cancer épithélial de l’ovaire (r = 0.445 et p= 0.00001), suggérant que ce paramètre pourrait servir comme marqueur tumoral. Afin de comprendre ces résultats, nous avons tout d’abord étudiés l’influence des mutations HFE sur les cellules cancéreuses. Pour ce faire, la lignée du cancer de l’ovaire TOV-112D, homozygote pour la mutation C282Y, a été transfectée avec les vecteurs HFEwt et HFEC282Y. Bien qu’aucune différence significative n’ait été trouvée en termes de TfR totaux, des analyses par FACS ont démontrées un phénotype de déficience de fer pour les clones stables HFEwt. In vitro, la restauration de la protéine HFE, dans la lignée TOV-112D du cancer de l’ovaire, n’influence pas la croissance cellulaire. Ensuite, nous avons étudiés l’influence des niveaux de fer sur la progression tumorale. Une expérience in vivo préliminaire a démontré une tendance à un volume tumoral supérieur dans un modèle de souris de surcharge de fer,HfeRag1-/-. De plus, les souris HfeRag1-/-, injectées avec la lignée du cancer de l’ovaire TOV-21G, ont montrées des niveaux significativement plus faibles de fer sérique comparativement à leur contrôle (fer sérique 40±7μM versus 27±6μM, p = 0.001). En conclusion, des études supplémentaires sont nécessaires afin de comprendre davantage le rôle des mutations HFE sur la progression tumorale. Notamment, les niveaux élevés de fer pourraient rendre les cellules tumorales résistantes aux traitements ou encore, augmenter la toxicité et ainsi, contribuer à un mauvais prognostique. / Epithelial ovarian cancer is the most aggressive gynecological cancer with the highest mortality rates. Growth of the ovarian cancer cells is limited by nutrients in the environment; iron being one of the elements essential to their proliferation. Hereditary hemochromatosis is a disease associated with an accumulation of body iron, and is linked to two mutations of the HFE gene including C282Y and H63D. Given the influence of HFE protein on the entry of iron in the cell, mutations in the HFE gene may be associated with rapid growth of cancer cells. By genotyping the HFE gene of 526 patients with epithelial ovarian cancer, we have found that the allelic frequency of the C282Y mutation is significantly higher in patients with ovarian cancer compared to patients in the control group (5.9% versus 1.3% p = 0.02). Moreover, the 2-year survival rate, of patients with C282Y mutations and G3 ovarian tumor, is low (20%) when compared to patients without mutations (60%, p = 0.005). A multivariate survival analysis, using Cox’s regression model, also showed a hazard ratio value of 3.1,suggesting that patients with the C282Y mutation are 3 times more likely to have a poor survival (p =0.001). As well, correlation studies have demonstrated that serum ferritin levels were higher in patients with advanced grade of ovarian cancer (r = 0.445 and p = 0.00001), suggesting that this iron parameter could serve as a tumor marker for assessing the progression of ovarian cancer. In order to investigate these findings, we first studied the influence of HFE mutations on cancer cells. The ovarian cancer cell line TOV-112D, homozygous for the C282Y mutation, was transfected with the HFEwt and HFEC282Y vector, and although there were no differences in total TfR levels, FACS analysis demonstrated an iron deficient phenotype for the HFEwt stable clones (p<0.05). In vitro,restoration of the HFE protein, in the TOV-112D ovarian cancer cell line, does not influence cell growth. We then studied the influence of iron levels on tumor progression. A preliminary in vivo experiment has demonstrated a tendency to a higher tumor volume in a mouse model of iron overload,HfeRag1-/-. Furthermore, HfeRag1-/- mice that were injected with the ovarian cancer cell line TOV-21G showed significant lower serum iron levels compared to their controls (SI 40±7μM versus 27±6μM, p = 0.001). In conclusion, more studies are required to further understand the role of HFE mutations on tumor progression. Higher iron levels may confer tumor cells to be drug resistant or increase toxicity and thus, may contribute to a bad prognostic.

Le cancer épithélial de l’ovaire est le cancer gynécologique le plus agressif avec le plus haut taux de mortalité. La croissance des cellules cancéreuses de l’ovaire est limitée par les nutriments de l’environnement, le fer étant un des éléments indispensables à leur prolifération. L’hémochromatose héréditaire est une maladie associée à une accumulation corporelle de fer. Cette maladie est liée à deux mutations majeures du gène HFE soit H63D et C282Y. Étant donnée l’influence de la protéine HFE sur l’entrée du fer dans la cellule, des mutations du gène HFE pourraient être associées à une croissance rapide des cellules cancéreuses. Des études de génotypage du gène HFE effectuées chez 526 patientes avec cancer épithélial de l’ovaire, ont révélées une fréquence allélique de la mutation C282Y significativement plus élevées chez les patientes avec tumeur ovarienne comparativement aux patientes du groupe contrôle (5.9% versus 1.3%, p = 0.02). De plus, le taux de survie des patientes avec mutations C282Y et tumeur ovarienne de G3, après 2 ans, est faible (20%) lorsque comparé à celui des patientes sans mutations (60%, p = 0.005). Une analyse de régression multivariée de Cox a démontrée un risque relatif de 3.1, suggérant que les patientes avec mutations C282Y ont 3 fois plus de chance d’avoir une faible survie (p=0.001). Également, des études de corrélation ont démontrées que les niveaux de ferritine du sérum étaient plus élevés chez les patientes avec grade avancé du cancer épithélial de l’ovaire (r = 0.445 et p= 0.00001), suggérant que ce paramètre pourrait servir comme marqueur tumoral. Afin de comprendre ces résultats, nous avons tout d’abord étudiés l’influence des mutations HFE sur les cellules cancéreuses. Pour ce faire, la lignée du cancer de l’ovaire TOV-112D, homozygote pour la mutation C282Y, a été transfectée avec les vecteurs HFEwt et HFEC282Y. Bien qu’aucune différence significative n’ait été trouvée en termes de TfR totaux, des analyses par FACS ont démontrées un phénotype de déficience de fer pour les clones stables HFEwt. In vitro, la restauration de la protéine HFE, dans la lignée TOV-112D du cancer de l’ovaire, n’influence pas la croissance cellulaire. Ensuite, nous avons étudiés l’influence des niveaux de fer sur la progression tumorale. Une expérience in vivo préliminaire a démontré une tendance à un volume tumoral supérieur dans un modèle de souris de surcharge de fer,HfeRag1-/-. De plus, les souris HfeRag1-/-, injectées avec la lignée du cancer de l’ovaire TOV-21G, ont montrées des niveaux significativement plus faibles de fer sérique comparativement à leur contrôle (fer sérique 40±7μM versus 27±6μM, p = 0.001). En conclusion, des études supplémentaires sont nécessaires afin de comprendre davantage le rôle des mutations HFE sur la progression tumorale. Notamment, les niveaux élevés de fer pourraient rendre les cellules tumorales résistantes aux traitements ou encore, augmenter la toxicité et ainsi, contribuer à un mauvais prognostique. / Epithelial ovarian cancer is the most aggressive gynecological cancer with the highest mortality rates. Growth of the ovarian cancer cells is limited by nutrients in the environment; iron being one of the elements essential to their proliferation. Hereditary hemochromatosis is a disease associated with an accumulation of body iron, and is linked to two mutations of the HFE gene including C282Y and H63D. Given the influence of HFE protein on the entry of iron in the cell, mutations in the HFE gene may be associated with rapid growth of cancer cells. By genotyping the HFE gene of 526 patients with epithelial ovarian cancer, we have found that the allelic frequency of the C282Y mutation is significantly higher in patients with ovarian cancer compared to patients in the control group (5.9% versus 1.3% p = 0.02). Moreover, the 2-year survival rate, of patients with C282Y mutations and G3 ovarian tumor, is low (20%) when compared to patients without mutations (60%, p = 0.005). A multivariate survival analysis, using Cox’s regression model, also showed a hazard ratio value of 3.1,suggesting that patients with the C282Y mutation are 3 times more likely to have a poor survival (p =0.001). As well, correlation studies have demonstrated that serum ferritin levels were higher in patients with advanced grade of ovarian cancer (r = 0.445 and p = 0.00001), suggesting that this iron parameter could serve as a tumor marker for assessing the progression of ovarian cancer. In order to investigate these findings, we first studied the influence of HFE mutations on cancer cells. The ovarian cancer cell line TOV-112D, homozygous for the C282Y mutation, was transfected with the HFEwt and HFEC282Y vector, and although there were no differences in total TfR levels, FACS analysis demonstrated an iron deficient phenotype for the HFEwt stable clones (p<0.05). In vitro,restoration of the HFE protein, in the TOV-112D ovarian cancer cell line, does not influence cell growth. We then studied the influence of iron levels on tumor progression. A preliminary in vivo experiment has demonstrated a tendency to a higher tumor volume in a mouse model of iron overload,HfeRag1-/-. Furthermore, HfeRag1-/- mice that were injected with the ovarian cancer cell line TOV-21G showed significant lower serum iron levels compared to their controls (SI 40±7μM versus 27±6μM, p = 0.001). In conclusion, more studies are required to further understand the role of HFE mutations on tumor progression. Higher iron levels may confer tumor cells to be drug resistant or increase toxicity and thus, may contribute to a bad prognostic.

[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.