Background: Lactose intolerance (LI) is an inability or reduced ability to produce the enzyme lactase. This disorder causes discomfort like stomach pain and/or diarrhea. The most common cause of LI for adults is a mutation in the MCM6 gene, a single nucleotide polymorphism at C/T-13910. Haemochromatosis (HH) is a hereditary disease that causes more iron uptake from food than normal, which leads to more iron circulating in the blood and causing more iron to be stored in the liver. About 90% of HH patients are homozygous for C282Y and there is a connection between compound heterozygous for C282Y and H63D and potential development of HH. In this study, two different methods were used and compared to analyze the gene for LI (LCT) and the gene for HH (HFE). An extraction-free method by LaCAR and a DNA extraction In-house method done at Sundsvalls hospital Sweden. Method: Genotyping for LCT and HFE was done by polymerase chain reaction (PCR) technique on DNA extracted from whole blood (EDTA samples). Results: All the blood samples gave the same genotype on both methods. One LI sample had an extra curve at T/G-13915, which was detected by the LaCAR method but not the in-house method. One HH sample had a different C curve at 187, which was detected by both methods. Conclusion: The LaCAR method was identified as a good alternative for the analysis of LCT and HFE genotypes. It was able to detect additional mutations in the MCM6 gene for LCT analysis and has a shorter run time.

A doença hepática associada a sobrecarga de ferro pode ocorrer devido a causas genéticas ou secundárias. Esse estudo avaliou pacientes com hepatopatia crônica com sobrecarga de ferro submetidos à pesquisa das mutações HFE no período de 2007-2009 e classificou como portadores de hemocromatose hereditária HFE (HH-HFE) aqueles que apresentavam as mutações C282Y/C282Y ou C282Y/H63D e como sobrecarga de ferro não HFE aqueles que apresentavam outras mutações no gene HFE como C282Y/-, H63D/- e H63D/H63D ou pacientes sem qualquer uma dessas mutações mencionadas. Os objetivos do estudo foram 1) analisar e correlacionar os aspectos fenotípicos e genotípicos de grupo de indivíduos com doença hepática crônica e sobrecarga de ferro; 2) caracterizar o quadro clínico, laboratorial e anatomopatológico, em busca de achados compatíveis com o fenótipo de hemocromatose; 3) Correlacionar o quadro clínico com as mutações no gene HFE. Foram analisados 108 indivíduos portadores de hepatopatia crônica selecionados a partir de saturação de transferrina (ST) > 45% e ferritina sérica > 350 ng/mL. Foram estudados e comparados 16 pacientes no grupo HH-HFE com 92 no grupo sobrecarga de ferro não HFE. Da casuística geral, a idade média ao diagnóstico da doença foi de 46,69 anos (16-77), com 70,73% constituída por indivíduos de cor branca, 77,57% do sexo masculino e 64,8% tinham cirrose hepática. A frequência de cirrose hepática não diferiu entre os grupos, entretanto, artropatia, carcinoma hepatocelular, diabetes e osteoporose foram mais frequentes no grupo HH- HFE (53,8% x 15,9%, 31,2% x 7,06%, 56,2% x 30%, 72,7% x 32,1%, respectivamente, p < 0,05). Os pacientes com mutações HFE diagnósticas de HH apresentaram maior chance de ter carcinoma hepatocelular (OR= 5,0, p= 0,032) quando comparados com os portadores de outros genótipos HFE e aqueles sem mutação. Os níveis de ST, ferro e ferritina também foram maiores naquele grupo, bem como os graus de siderose 3 e 4 (p= 0,026). A ST foi a variável que se correlacionou independentemente com o diagnóstico das mutações C282Y/C282Y e C282Y/H63D. A frequência de fatores de risco para sobrecarga de ferro não diferiu entre os grupos. Observou-se, entretanto, que no grupo HH-HFE havia maior número de pacientes sem qualquer fator de risco detectado (p= 0,019). Níveis de ST > 82% apresentaram maior valor preditivo negativo para o diagnóstico de HH-HFE do que os de ferritina, ferro, capacidade total de ligação de ferro e de transferrina. Concluímos que os portadores de HH-HFE têm maiores graus de sobrecarga de ferro quando comparados ao grupo de sobrecarga de ferro não-HFE; em indivíduos com doença hepática crônica. ST > 82% tem maior acurácia para diagnóstico de HH-HFE; portadores de mutações C282Y em homozigose ou em heterozigose composta com H63D têm maior chance de apresentar carcinoma hepatocelular do que os portadores de outras mutações no gene HFE e pacientes sem mutação / Chronic liver disease related to iron overload may occur due to genetic or secondary causes. This study analyzed patients with chronic liver diseases and iron overload who were tested for HFE mutations from 2007 to 2009. Patients with C282Y/C282Y or C282Y/H63D mutations were diagnosed with HFE hereditary hemochromatosis (HFE-HH) and those with other HFE genotypes (C282Y/-, H63D/- or H63D/H63D) or individuals without HFE mutations (wild type) were designed as non-HFE iron overload. The aims of this study were: 1) to analyze and to establish correlations between phenotypic and genotypic aspects of individuals with chronic liver disease and with iron overload; 2) to charachterize the clinical manifestations, laboratory and histological findings consistent with the phenotype of hemochromatosis; 3) to verify associations between clinical manifestations and HFE mutations. One hundred and eight patients with chronic liver diseases and with iron overload, defined as transferrin saturation (TS) > 45% and serum ferritin levels > 350 ng/mL were included. Sixteen patients had HH-HFE and were compared with 92 patients with non-HFE iron overload group. The average of age at diagnosis was 46.69 years (16-77), 70.73% were Caucasians, 77.57% were male and 64.8% had hepatic cirrhosis. The proportion of hepatic cirrhosis was similar in both groups, nevertheless arthropathy, hepatocellular carcinoma, diabetes and osteoporosis were more frequent in the HFE-HH group (53,8% x 15,9%, 31,2% x 7,06%, 56,2% x 30%, 72,7% x 32,1%, respectively, p < 0,05). The HFE C282Y/C282Y or C282Y/ H63D genotypes had a higher chance to develop hepatocellular carcinoma (OR= 5.0, p= 0.032) when compared with the other HFE genotypes and with those wild type. The levels of TS, serum iron and ferritin were greater in HFE-HH group, as well as hepatic siderosis grade 3 and 4 (p= 0.026). TS was the biochemical marker of iron overload with the higher independent correlation with the presence of C282Y/C282Y and C282Y/H63D mutations. The frequency of risk factors for iron overload was not different between the groups, however, in HFE-HH group a greater number of patients without any risk factor was detected (p= 0.019). TS > 82% had a higher predictive negative value for diagnosing HFE-HH when compared to the levels of ferritin, serum iron, total iron binding capacity and transferrin. We concluded that the HFE-HH patients had a greater iron overload than patients with chronic liver diseases with non-HFE iron overload. TS > 82% had more accuracy to diagnose HFE-HH. The carriers of C282Y/C282Y or C282Y/H63D mutations had a higher probability to develop hepatocellular carcinoma, when compared to the patients with HFE genotypes and patients wild type

A doença hepática associada a sobrecarga de ferro pode ocorrer devido a causas genéticas ou secundárias. Esse estudo avaliou pacientes com hepatopatia crônica com sobrecarga de ferro submetidos à pesquisa das mutações HFE no período de 2007-2009 e classificou como portadores de hemocromatose hereditária HFE (HH-HFE) aqueles que apresentavam as mutações C282Y/C282Y ou C282Y/H63D e como sobrecarga de ferro não HFE aqueles que apresentavam outras mutações no gene HFE como C282Y/-, H63D/- e H63D/H63D ou pacientes sem qualquer uma dessas mutações mencionadas. Os objetivos do estudo foram 1) analisar e correlacionar os aspectos fenotípicos e genotípicos de grupo de indivíduos com doença hepática crônica e sobrecarga de ferro; 2) caracterizar o quadro clínico, laboratorial e anatomopatológico, em busca de achados compatíveis com o fenótipo de hemocromatose; 3) Correlacionar o quadro clínico com as mutações no gene HFE. Foram analisados 108 indivíduos portadores de hepatopatia crônica selecionados a partir de saturação de transferrina (ST) > 45% e ferritina sérica > 350 ng/mL. Foram estudados e comparados 16 pacientes no grupo HH-HFE com 92 no grupo sobrecarga de ferro não HFE. Da casuística geral, a idade média ao diagnóstico da doença foi de 46,69 anos (16-77), com 70,73% constituída por indivíduos de cor branca, 77,57% do sexo masculino e 64,8% tinham cirrose hepática. A frequência de cirrose hepática não diferiu entre os grupos, entretanto, artropatia, carcinoma hepatocelular, diabetes e osteoporose foram mais frequentes no grupo HH- HFE (53,8% x 15,9%, 31,2% x 7,06%, 56,2% x 30%, 72,7% x 32,1%, respectivamente, p < 0,05). Os pacientes com mutações HFE diagnósticas de HH apresentaram maior chance de ter carcinoma hepatocelular (OR= 5,0, p= 0,032) quando comparados com os portadores de outros genótipos HFE e aqueles sem mutação. Os níveis de ST, ferro e ferritina também foram maiores naquele grupo, bem como os graus de siderose 3 e 4 (p= 0,026). A ST foi a variável que se correlacionou independentemente com o diagnóstico das mutações C282Y/C282Y e C282Y/H63D. A frequência de fatores de risco para sobrecarga de ferro não diferiu entre os grupos. Observou-se, entretanto, que no grupo HH-HFE havia maior número de pacientes sem qualquer fator de risco detectado (p= 0,019). Níveis de ST > 82% apresentaram maior valor preditivo negativo para o diagnóstico de HH-HFE do que os de ferritina, ferro, capacidade total de ligação de ferro e de transferrina. Concluímos que os portadores de HH-HFE têm maiores graus de sobrecarga de ferro quando comparados ao grupo de sobrecarga de ferro não-HFE; em indivíduos com doença hepática crônica. ST > 82% tem maior acurácia para diagnóstico de HH-HFE; portadores de mutações C282Y em homozigose ou em heterozigose composta com H63D têm maior chance de apresentar carcinoma hepatocelular do que os portadores de outras mutações no gene HFE e pacientes sem mutação / Chronic liver disease related to iron overload may occur due to genetic or secondary causes. This study analyzed patients with chronic liver diseases and iron overload who were tested for HFE mutations from 2007 to 2009. Patients with C282Y/C282Y or C282Y/H63D mutations were diagnosed with HFE hereditary hemochromatosis (HFE-HH) and those with other HFE genotypes (C282Y/-, H63D/- or H63D/H63D) or individuals without HFE mutations (wild type) were designed as non-HFE iron overload. The aims of this study were: 1) to analyze and to establish correlations between phenotypic and genotypic aspects of individuals with chronic liver disease and with iron overload; 2) to charachterize the clinical manifestations, laboratory and histological findings consistent with the phenotype of hemochromatosis; 3) to verify associations between clinical manifestations and HFE mutations. One hundred and eight patients with chronic liver diseases and with iron overload, defined as transferrin saturation (TS) > 45% and serum ferritin levels > 350 ng/mL were included. Sixteen patients had HH-HFE and were compared with 92 patients with non-HFE iron overload group. The average of age at diagnosis was 46.69 years (16-77), 70.73% were Caucasians, 77.57% were male and 64.8% had hepatic cirrhosis. The proportion of hepatic cirrhosis was similar in both groups, nevertheless arthropathy, hepatocellular carcinoma, diabetes and osteoporosis were more frequent in the HFE-HH group (53,8% x 15,9%, 31,2% x 7,06%, 56,2% x 30%, 72,7% x 32,1%, respectively, p < 0,05). The HFE C282Y/C282Y or C282Y/ H63D genotypes had a higher chance to develop hepatocellular carcinoma (OR= 5.0, p= 0.032) when compared with the other HFE genotypes and with those wild type. The levels of TS, serum iron and ferritin were greater in HFE-HH group, as well as hepatic siderosis grade 3 and 4 (p= 0.026). TS was the biochemical marker of iron overload with the higher independent correlation with the presence of C282Y/C282Y and C282Y/H63D mutations. The frequency of risk factors for iron overload was not different between the groups, however, in HFE-HH group a greater number of patients without any risk factor was detected (p= 0.019). TS > 82% had a higher predictive negative value for diagnosing HFE-HH when compared to the levels of ferritin, serum iron, total iron binding capacity and transferrin. We concluded that the HFE-HH patients had a greater iron overload than patients with chronic liver diseases with non-HFE iron overload. TS > 82% had more accuracy to diagnose HFE-HH. The carriers of C282Y/C282Y or C282Y/H63D mutations had a higher probability to develop hepatocellular carcinoma, when compared to the patients with HFE genotypes and patients wild type

A hemocromatose hereditária é caracterizada pelo aumento da absorção intestinal de ferro, acarretando progressivo acúmulo no organismo. Os objetivos foram: 1- determinar as frequências das mutações p.C282Y, p.H63D e p.S65C no gene HFE e avaliar os efeitos nas concentrações dos parâmetros do ferro em doadores de sangue; 2- pesquisar mutações nos genes: 2.1- HFE, 2.2- HJV e HAMP, 2.3- TFR2 e SLC40A1, em pacientes portadores de sobrecarga de ferro primária. Participaram 542 doadores de sangue provenientes do Hemocentro da Santa Casa de São Paulo. Foram incluídos 51 pacientes que apresentavam saturação de transferrina &#8805; 50% (para mulheres) e &#8805; 60% (para homens) e ausência de causas secundárias. Os genótipos para as mutações nos genes HFE foram avaliados pela PCR-RFLP. Foi realizado sequenciamento direto bidirecional para cada éxon dos genes, utilizando o sequenciador Genetic Analizer 3500XL®. Nos doadores de sangue, as frequências dos alelos HFE 282Y, HFE 63D e HFE 65C foram 2,1, 13,6 e 0,6%, respectivamente. Os homens que doaram pela primeira vez, portadores do genótipo HFE 282CY, apresentaram maiores valores de saturação de transferrina; e também os portadores dos genótipos HFE 63DD e 63HD apresentaram maiores concentrações de ferritina sérica, em relação aos de genótipo selvagem. Para os pacientes, 72,5% (37/51) apresentaram ao menos 1 alteração no gene HFE e 11 foram identificados como homozigotos para a mutação p.C282Y. Uma mutação não descrita na literatura (p.V256I) foi identificada no gene HFE e a modelagem molecular (análises de ligação e estrutural) detectou que a mutação não reduziu a afinidade entre as proteínas HFE e &#946;2-microglobulina. No sequenciamento dos éxons dos genes HJV e HAMP foram identificadas as alterações já descritas: HJV p.E302K, HJV p.A310G, HJV p.G320V e HAMP p.R59G. Para o gene TFR2, foram identificados 3 polimorfismos já descritos (p.A75V, p.A617A e p.R752H). No gene SLC40A1 foram observados 6 polimorfismos (rs13008848, rs11568351, rs11568345, rs11568344, rs2304704 e rs11568346) e 1 alteração não descrita previamente na literatura (p.G204S). As conclusões foram: 1- em relação aos doadores de sangue, a presença dos alelos HFE 282Y e HFE 63D foi associada ao maior aporte de ferro nos homens que não doaram sangue anteriormente. 2.1- Para os pacientes com sobrecarga de ferro, a mutação p.C282Y em homozigose, ou em heterozigose composta com a p.H63D, foi a mais frequente alteração encontrada (33,3%). 2.2- O diagnóstico molecular de hemocromatose juvenil (HJ) no Brasil (HJV p.G320V em homozigose) foi relatado. As mutações funcionais HJV p.E302K e HAMP p.R59G foram identificadas, sendo possível que estas alterações possam estar contribuindo para consequências fenotípicas juntas as outras mutações em regiões intrônicas ou regulatórias dos genes. 2.3- Mutações funcionais nos genes TFR2 e SLC40A1 não foram identificadas. / Hereditary hemochromatosis (HH) is characterized by increased intestinal iron absorption, which leads to a progressive accumulation of iron in the body. The aims were: 1- to assess the frequency of HFE gene mutations (p.C282Y, p.H63D and p.S65C) and to identify their relationship to iron status in blood donors; 2- to search in primary iron overload patients: 2.1- HFE, 2.2- HJV and HAMP, 2.3- TFR2 and SLC40A1 gene mutations. Blood donors (n=542) were recruited from Hemocentro of Santa Casa Hospital, Sao Paulo, Brazil. The study included 51 patients with transferrin saturation &#8805; 50% (women) and &#8805; 60% (men) and absence of secondary causes. The genotypes for HFE mutations were evaluated by PCR-RFLP. Subsequent bidirectional sequencing for each gene was performed using the Genetic Analizer sequencer 3500XL®. The frequencies of HFE 282Y, HFE 63D and HFE 65C alleles were 2.1, 13.6 and 0.6% in blood donors, respectively. The first time male donors carrying heterozygous genotype for the p.C282Y mutation had higher transferrin saturation values; and men carrying HFE 63DD and 63HD genotypes had higher serum ferritin concentrations when compared to the wild genotype. Thirtyseven (72.5%) out of the 51 patients presented at least one HFE mutation and 11 were identified as homozygous for the mutation p.C282Y. One novel mutation (p.V256I) in the HFE gene was indentified and molecular modeling (free energy and structural analysis in silico) showed that p.V256I mutation did not reduce the affinity binding between HFE and &#946;2-microglobulin. Sequencing in the HJV and HAMP genes revealed HJV p.E302K, HJV p.A310G, HJV p.G320V and HAMP p.R59G alterations. Sequencing in the TFR2 gene observed 3 polymorphisms (p.A75V, p.A617A e p.R752H); and sequencing in the SLC40A1 gene identified 6 polymorphisms (rs13008848, rs11568351, rs11568345, rs11568344, rs2304704 e rs11568346) and 1 p.G204S non-described mutation. The conclusions were: 1- for blood donors, the presence of HFE 282Y and HFE 63D alleles were associated with alterations on iron status only in first time male blood donors. 2.1- For patients with iron overload, the p.C282Y mutation in homozygous or in compound heterozygous with p.H63D, was the most frequent molecular change found (33.3%). 2.2- The molecular diagnosis of Juvenil Hemochromatosis (JH) in Brazil (homozygous genotype for the HJV p.G320V) was reported. The HJV p.E302K and HAMP p.R59G functional mutations were found and, it is conceivable that they may be contributing to phenotypic consequences together to other mutations in intronic or regulatory regions. 2.3- Functional mutation in the TFR2 and SLC40A1 genes were not identified.

[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.

Thesis (MSc (Genetics))—University of Stellenbosch, 2007. / Hereditary haemochromatosis (HH), a common autosomal recessive disease, is characterized by increased iron absorption leading to progressive iron accumulation in organs such as the liver, heart and pancreas. In the South African population the disease is prevalent in individuals of Caucasian origin, with a carrier frequency of one in six for the C282Y mutation in the HFE gene. We investigated the role of genes implicated in iron metabolism, including the high-iron gene (HFE), haem oxgenase-1 gene (HMOX1), solute carrier family 40 (iron-regulated transporter) member 1 gene (SLC40A1), cytochrome b reductase gene (CYBRD1), hepcidin antimicrobial peptide gene (HAMP) and the hemojuvelin gene (HJV) in a patient cohort with non-HFE iron overload. DNA analysis was performed on samples from 36 unrelated South African Caucasian patients presenting with primary iron overload, who tested either negative or heterozygous for C282Y. In this study, mutation screening was performed by PCR amplification and HEX-SSCP analysis. Sixteen previously described and two novel variants were identified by semi-automated DNA sequencing. Common variants identified in the HFE gene included C282Y, H63D, IVS2+4T→C, IVS4-44T→C, IVS4+48G→A and IVS5-47G→A. The Q127H mutation in exon 3 of the HFE gene was identified in one patient, who tested negative for both C282Y and H63D. Mutation S65C was identified only in the population-matched controls and was absent in the patient group. Other previously described polymorphisms identified included the IVS5+51delTGGCTGTCTGACT deletion in HMOX1, I109 and V221 in SLC40A1, IVS1-4C→G, IVS2+8T→C and S266N, in the CYBRD1 gene and, S264 and A310G in the HJV gene. The novel variants, -89C→T, in the promoter region of the CYBRD1 gene, was detected in only one patient, while S333 in exon 4 of the HJV gene was present in three patients. These variants were not identified in any of the population-matched controls screened and could explain the non-HFE iron overload presented by these patients. This study clearly demonstrates the importance of modifier genes in patients with iron overload that cannot be explained by the common C282Y mutation. Studies on iron-related genes and the identification of mutations in these genes in non-HFE patients could lead to improved diagnosis and counselling of South African patients presenting with primary iron overload.

La sclérose latérale amyotrophique (SLA) est une maladie neurodégénérative d’évolution toujours fatale. Dans les formes sporadiques (SLAS), l’étiologie demeure inconnue et l’hypothèse d’une participation génétique dans le cadre d’un modèle de maladie complexe est envisagée. Notre travail porte sur deux gènes de susceptibilité dans la SLAS : le gène de l’Apolipoprotéine E (APOE) et le gène impliqué dans l’hémochromatose familiale de type 1 (HFE). Notre première étude confirme sur une population de 1482 patients atteints de SLA sporadique le lien entre allèle ε4 et la forme bulbaire de la maladie mais uniquement chez les hommes. Nous proposons une explication physiopathologique faisant intervenir le récepteur aux androgènes, particulièrement exprimé au niveau des motoneurones bulbaires. Notre deuxième étude sur 244 patients et 302 contrôles ne retrouve pas l’association entre le polymorphisme H63D et le risque de SLA sporadique notée dans d’autres populations. En revanche, l’allèle Y du polymorphisme C282Y semble exercer un effet protecteur vis-à-vis de la SLA. Ces données sont discutées dans le contexte de l’hypothèse physiopathologique du stress oxydant. / Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease. The cause of sporadic cases (SALS) remains unknown but a genetic participation in a model of complex disease is suspected. Our work concerns two susceptibility genes for SALS: Apolipoprotein E gene (APOE) and the gene involved in familial hemochromatosis (HFE). Our first study including 1482 patients with SALS confirms a link between ε4 allele and bulbar-onset of the disease, only in men. We suggest a pathophysiological explanation with a role for the androgen receptor which is particularly abundant in motor neurons of the brainstem. Our second study about 244 patients and 302 controls did not find any association between the H63D polymorphism and SLAS, which has previously been showed. However, the Y allele of the C282Y polymorphism seems to exert a protective effect against SALS. We discuss these data within the pathophysiological hypothesis of oxidative stress in ALS.

Made available in DSpace on 2014-06-11T19:26:03Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-02-27Bitstream added on 2014-06-13T20:54:00Z : No. of bitstreams: 1 estevao_if_me_sjrp.pdf: 1151592 bytes, checksum: 9e2d3a0a29b1ad6405857d13d891a9f1 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A beta talassemia é um dos mais freqüentes distúrbios genéticos no mundo. Estima-se que 1,5% a 3% da população mundial seja portadora do traço talassêmico. Esses portadores geralmente são oligo ou assintomáticos e têm uma expectativa de vida semelhante à dos não portadores. Entretanto, níveis elevados de ferritina sérica têm sido observados em alguns estudos comparativos entre beta talassemia heterozigota e não portadores e, alguns indivíduos, que nunca foram transfundidos, apresentam sinais clínicos e laboratoriais de sobrecarga de ferro. A fisiopatologia dessa complicação continua em discussão. Vários pesquisadores têm sugerido um efeito modulador da mutação do gene da beta globina e mutações em genes codificadores de proteínas relacionadas ao metabolismo do ferro. Mutações no gene HFE são as mais freqüentemente associadas à hemocromatose hereditária. O objetivo do presente trabalho foi avaliar a freqüência das mutações C282Y e H63D no gene HFE em portadores de beta talassemia heterozigota e analisar sua influência no metabolismo do ferro. Foram estudados 162 portadores de beta talassemia heterozigota, residentes na cidade de São Carlos ou região, caucasóides e, acompanhados no serviço de Hematologia. O diagnóstico de traço talassêmico foi confirmado em todos por meio do eritrograma e da quantificação da Hb A2 e Hb fetal por HPLC. O metabolismo do ferro foi avaliado pelas dosagens de ferro sérico, capacidade total de ligação do ferro, ferritina e saturação da transferrina e, a análise molecular das mutações no gene HFE, pela técnica de PCR-RLFP. Foram realizadas análises de correlação linear de Pearson por idade e gênero entre hemoglobina... / Beta thalassemia is one of the most frequent genetic disorder in the world. It is estimated that 1.5% to 3% of the world population is a thalassemia carrier. These individuals are generally slightly symptomatic or asymptomatic and they have a life expectancy similar to those who are non-carriers. However, high levels of serum ferritin have been observed in some comparative studies between heterozygous for beta thalassemia and non-carriers, and some individuals that were never transfused, present clinic and laboratories signs of iron overload. The physiopathology of this disease continues in discussion. Several researchers have suggested a modulator effect from the mutation of the beta globin gene and mutations in genes related with the iron metabolism. Mutations of the gene HFE are the most frequently associated to the hereditary hemochromatosis. The aim of this study was evaluate the frequency of C282Y and H63D mutations in the HFE gene in beta thalassemia carriers, and analyze its influence in the iron metabolism. 162 beta thalassemia carriers, Caucasoid, residing in the city of Sao Carlos or region and accompanied in the Hematology service were studied. The diagnostic of thalassemia trait was confirmed in every one through a complete erythrogram and quantification of Hb A2 and Hb fetal by HPLC. The iron metabolism was evaluated by serum iron, total iron-binding capacity, serum ferritin and percent saturation of transferring. The molecular analysis of the mutations in the HFE gene was made by PCR-RLFP. There were made analysis of linear Pearson’ correlation, by age and gender, among hemoglobin, Hb A2, VCM and among reticulocytes count and the values of saturation of transferrin and serum ferritin.

Made available in DSpace on 2014-06-11T19:32:14Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-02-27Bitstream added on 2014-06-13T21:03:49Z : No. of bitstreams: 1 baracioli_lmsv_dr_sjrp.pdf: 2904875 bytes, checksum: 978d5a56c7fb480e1bbe8b2353736483 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Ministério da Saúde / Universidade Estadual Paulista (UNESP) / As hemoglobinopatias são as mais comuns das doenças genéticas e são classificadas em Talassemias e Variantes de Hemoglobinas. A Hb S é originada por uma mutação de ponto no códon que origina o aminoácido da posição seis da cadeia beta globina, resultando em uma modificação estrutural na molécula que altera sua solubilidade e cristalização. A Hb S apresenta efeito sobre as células sangüíneas, desencadeando processos oxidativos com geração de radicais livres. Efeito semelhante observa-se no acúmulo de ferro, em decorrência da hemocromatose hereditária. Os defeitos de metabolismo do ferro, em especial aqueles oriundos dos polimorfismos no gene HFE, levam ao acúmulo de ferro tecidual, com potencial capacidade oxidante e peroxidação lipídica. Fundamentados no possível efeito tóxico da mutação no gene da globina βS (Hb S) e do acúmulo de ferro tecidual, relatados na literatura, objetivou-se avaliar a influência dos polimorfismos do gene HFE na peroxidação lipídica dos portadores dessa alteração de hemoglobina e sua relação com o aumento da destruição eritrocitária e geração de estresse oxidativo. Foram analisadas 16.000 amostras de doadores de sangue e encontrou-se uma freqüência de Hb AS de 1,69% na região Noroeste do Estado de São Paulo. Destes, 43 apresentaram herança da Hb S e de pelo menos um dos dois polimorfismos no gene HFE testados. A freqüência dos polimorfismos C282Y e H63D, no gene HFE, no grupo de doadores com Hb AS, esta de acordo com os dados da literatura. No grupo de doadores de sangue com Hb AS observamos que, na herança concomitante dos dois polimorfismos, os níveis de MDA, indicativo de peroxidação lipídica, estavam elevados, indicando a influência de ambas afecções genéticas, no aumento dos níveis de peroxidação lipídica. Entretanto, os níveis médios da TEAC, indicativo da capacidade oxidante, apresentaram-se... / The hemoglobinopathies are the most common of the genetic disease and are classified in Thalassemia and Hemoglobin Variants. Hb S is originated by a point mutation in the codon that originates the sixth aminoacid of the β globin chain, resulting in a structural modification in the molecule that modifies the solubility and crystallization. Hb S presents an effect on the blood cells, unchaining the oxidative processes with the free radicals generation. Similar effect is observed in the iron accumulation, in result of the hereditary hemochromatosis. The iron metabolism defects, in special those one from the HFE gene polymorphism, lead to the tissue iron accumulation with potential oxidant capacity and lipid peroxidation. Based on the possible toxic effect of the mutation in the globin βS gene (Hb S) and tissue iron accumulation, mentioned in the literature, we was aimed evaluate the influence of HFE gene polymorphism in the lipid peroxidation in blood donors with hemoglobin S alteration and the relation with the eritrocitary destruction increase and oxidative stress generation. 16,000 samples of blood donor had been analyzed and it was found a Hb frequency of 1,69% from the Northwest São Paulo state region. Among them, 43 had presented concomitance in the Hb S inheritance and one of the two HFE gene polymorphism. The frequency of C282Y and H63D polymorphism, in the HFE gene, in the blood donor group with Hb S, is in according to the literature. We also verified in the blood donor group with Hb AS that, in the concomitant inheritance of the two polymorphism, the MDA levels, indicative of lipid peroxidation, were raised, showing the influence of both genetic affections, in the levels of lipid peroxidation. However, the TEAC average levels, indicative of the oxidant capacity, had been presented normal, indicating that in spite of a lipid peroxidation raise, this is not followed... (Complete abstract click electronic access below)

Orientador: Claudia Regina Bonini Domingos / Banca: Rodolfo Delfini Cançado / Banca: Ivan de Lucena Angulo / Banca: Maria Tercilia de Azeredo Oliveira / Banca: Eduardo Alves de Almeida / Resumo: As hemoglobinopatias são as mais comuns das doenças genéticas e são classificadas em Talassemias e Variantes de Hemoglobinas. A Hb S é originada por uma mutação de ponto no códon que origina o aminoácido da posição seis da cadeia beta globina, resultando em uma modificação estrutural na molécula que altera sua solubilidade e cristalização. A Hb S apresenta efeito sobre as células sangüíneas, desencadeando processos oxidativos com geração de radicais livres. Efeito semelhante observa-se no acúmulo de ferro, em decorrência da hemocromatose hereditária. Os defeitos de metabolismo do ferro, em especial aqueles oriundos dos polimorfismos no gene HFE, levam ao acúmulo de ferro tecidual, com potencial capacidade oxidante e peroxidação lipídica. Fundamentados no possível efeito tóxico da mutação no gene da globina βS (Hb S) e do acúmulo de ferro tecidual, relatados na literatura, objetivou-se avaliar a influência dos polimorfismos do gene HFE na peroxidação lipídica dos portadores dessa alteração de hemoglobina e sua relação com o aumento da destruição eritrocitária e geração de estresse oxidativo. Foram analisadas 16.000 amostras de doadores de sangue e encontrou-se uma freqüência de Hb AS de 1,69% na região Noroeste do Estado de São Paulo. Destes, 43 apresentaram herança da Hb S e de pelo menos um dos dois polimorfismos no gene HFE testados. A freqüência dos polimorfismos C282Y e H63D, no gene HFE, no grupo de doadores com Hb AS, esta de acordo com os dados da literatura. No grupo de doadores de sangue com Hb AS observamos que, na herança concomitante dos dois polimorfismos, os níveis de MDA, indicativo de peroxidação lipídica, estavam elevados, indicando a influência de ambas afecções genéticas, no aumento dos níveis de peroxidação lipídica. Entretanto, os níveis médios da TEAC, indicativo da capacidade oxidante, apresentaram-se... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The hemoglobinopathies are the most common of the genetic disease and are classified in Thalassemia and Hemoglobin Variants. Hb S is originated by a point mutation in the codon that originates the sixth aminoacid of the β globin chain, resulting in a structural modification in the molecule that modifies the solubility and crystallization. Hb S presents an effect on the blood cells, unchaining the oxidative processes with the free radicals generation. Similar effect is observed in the iron accumulation, in result of the hereditary hemochromatosis. The iron metabolism defects, in special those one from the HFE gene polymorphism, lead to the tissue iron accumulation with potential oxidant capacity and lipid peroxidation. Based on the possible toxic effect of the mutation in the globin βS gene (Hb S) and tissue iron accumulation, mentioned in the literature, we was aimed evaluate the influence of HFE gene polymorphism in the lipid peroxidation in blood donors with hemoglobin S alteration and the relation with the eritrocitary destruction increase and oxidative stress generation. 16,000 samples of blood donor had been analyzed and it was found a Hb frequency of 1,69% from the Northwest São Paulo state region. Among them, 43 had presented concomitance in the Hb S inheritance and one of the two HFE gene polymorphism. The frequency of C282Y and H63D polymorphism, in the HFE gene, in the blood donor group with Hb S, is in according to the literature. We also verified in the blood donor group with Hb AS that, in the concomitant inheritance of the two polymorphism, the MDA levels, indicative of lipid peroxidation, were raised, showing the influence of both genetic affections, in the levels of lipid peroxidation. However, the TEAC average levels, indicative of the oxidant capacity, had been presented normal, indicating that in spite of a lipid peroxidation raise, this is not followed... (Complete abstract click electronic access below) / Doutor