Endothelial function, antioxidant status and vascular compliance in newly diagnosed HFE C282Y homozygotes

Purpose: This pilot study was aimed to establish techniques for assessing and observing trends in endothelial function, antioxidant status and vascular compliance in newly diagnosed HFE haemochromatosis during the first year of venesection.

Patients/methods: Untreated newly diagnosed HFE haemochromatosis patients were tested for baseline liver function, iron indices, lipid profile, markers of endothelial function, anti-oxidant status and vascular compliance. Following baseline assessment, subjects attended at 6-weeks and at 3, 6, 9 and 12-months for follow-up studies.

Results: Ten patients were recruited (M = 8, F = 2, mean age = 51 years). Venesection significantly increased high density lipoproteins at 12-months (1.25 mmol/L vs. 1.37 mmol/L, p = 0.01). However, venesection did not significantly affect lipid hydroperoxides, intracellular and vascular cell adhesion molecules or high sensitivity C-reactive protein (0.57 mu mol/L vs. 0.51 mu mol/L, p = 0.45, 427.4 ng/ml vs. 307.22 ng/ml, p = 0.54, 517.70 ng/ml vs. 377.50 ng/ml, p = 0.51 and 290.75 mu g/dL vs. 224.26 mu g/dL, p = 0.25). There was also no significant effect of venesection on anti-oxidant status or pulse wave velocity (9.65 m/s vs. 8.74 m/s, p = 0.34).

Conclusions: Venesection significantly reduced high density lipoproteins but was not associated with significant changes in endothelial function, anti-oxidant status or vascular compliance. Larger studies using this established methodology are required to clarify this relationship further. 

Effect of C282Y Genotype on Self-Reported Musculoskeletal Complications in Hereditary Hemochromatosis

OBJECTIVE: Arthropathy that mimics osteoarthritis (OA) and osteoporosis (OP) is considered a complication of hereditary hemochromatosis (HH). We have limited data comparing OA and OP prevalence among HH patients with different hemochromatosis type 1 (HFE) genotypes. We investigated the prevalence of OA and OP in patients with HH by C282Y homozygosity and compound heterozygosity (C282Y/H63D) genotype. METHODS: A total of 306 patients with HH completed a questionnaire. Clinical and demographic characteristics and presence of OA, OP and related complications were compared by genotype, adjusting for age, sex, body mass index (BMI), current smoking and menopausal status. RESULTS: In total, 266 of the 306 patients (87%) were homozygous for C282Y, and 40 (13%) were compound heterozygous. The 2 groups did not differ by median age [60 (interquartile range [IQR] 53 to 68) vs. 61 (55 to 67) years, P=0.8], sex (female: 48.8% vs. 37.5%, P=0.18) or current smoking habits (12.4% vs. 10%, P=0.3). As compared with compound heterozygous patients, C282Y homozygous patients had higher median serum ferritin concentration at diagnosis [1090 (IQR 610 to 2210) vs. 603 (362 to 950) µg/L, P<0.001], higher median transferrin saturation [80% (IQR 66 to 91%) vs. 63% (55 to 72%), P<0.001]) and lower median BMI [24.8 (22.1 to 26.9) vs. 26.2 (23.5 to 30.3) kg/m2, P<0.003]. The overall prevalence of self-reported OA was significantly higher with C282Y homozygosity than compound heterozygosity (53.4% vs. 32.5%; adjusted odds ratio [aOR] 2.4 [95% confidence interval 1.2-5.0]), as was self-reported OP (25.6% vs. 7.5%; aOR 3.5 [1.1-12.1]). CONCLUSION: Patients with C282Y homozygosity may be at increased risk of musculoskeletal complications of HH.

Análise das mutações C282Y e H63D no gene da proteína HFE em pacientes com hiperferritinemia

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

Frequency of the HFE C282Y and H63D polymorphisms in Brazilian malaria patients and blood donors from the Amazon region

Malaria is an endemic parasitosis and its causitive agent, Plasmodium, has a metabolism linked to iron supply. HFE is a gene with the polymorphisms C282Y and H63D, which are associated with a progressive iron accumulation in the organism leading to a disease called hereditary hemochromatosis. The aim of the present study was to determine the allelic and genotypic frequencies of the HFE gene polymorphisms in malaria patients and blood donors from the Brazilian Amazon region. We screened 400 blood donors and 400 malaria patients for the HFE C282Y and H63D polymorphisms from four states of the Brazilian Amazon region by polymerase chain reaction and restriction fragment length polymorphism analysis. We did not find any C282Y homozygous individuals, and the only five heterozygous individuals detected were from Pará State. The most frequent genotype in the North region of Brazil was the H63D heterozygote, in both study groups. Our results contribute to the concept that the Brazilian Amazon region should not be regarded as a single entity in South America. These polymorphisms did not influence the symptoms of malaria in the population studied, as neither severe signs nor high parasitemia were observed. Therefore, different hereditary hemochromatosis diagnostic and control measures must be developed and applied within its diverse locations. Investigations are currently being carried out in our laboratory in order to determine the importance of the coexistence of hereditary hemochromatosis in patients affected by parasitic diseases, such as malaria. ©FUNPEC-RP.

Frequência dos mutantes C282Y e H63D do gene HFE e sua influência no metabolismo do ferro e na expressão da beta talassemia heterozigota

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Hereditary hemochromatosis: Effects of C282Y and H63D mutations on association with β2-microglobulin, intracellular processing, and cell surface expression of the HFE protein in COS-7 cells

Hereditary hemochromatosis (HH) is the most common autosomal recessive disorder known in humans. A candidate gene for HH called HFE has recently been cloned that encodes a novel member of the major histocompatibility complex class I family. Most HH patients are homozygous for a Cys-282→Tyr (C282Y) mutation in HFE gene, which has been shown to disrupt interaction with β2-microglobulin; a second mutation, His-63→Asp (H63D), is enriched in HH patients who are heterozygous for C282Y mutation. The aims of this study were to determine the effects of the C282Y and H63D mutations on the cellular trafficking and degradation of the HFE protein in transfected COS-7 cells. The results indicate that, while the wild-type and H63D HFE proteins associate with β2-microglobulin and are expressed on the cell surface of COS-7 cells, these capabilities are lost by the C282Y HFE protein. We present biochemical and immunofluorescence data that indicate that the C282Y mutant protein: (i) is retained in the endoplasmic reticulum and middle Golgi compartment, (ii) fails to undergo late Golgi processing, and (iii) is subject to accelerated degradation. The block in intracellular transport, accelerated turnover, and failure of the C282Y protein to be presented normally on the cell surface provide a possible basis for impaired function of this mutant protein in HH.

Increased C282Y heterozygosity in gestational diabetes

Background. Hereditary hemochromatosis is an autosomal recessive disorder of iron metabolism that is characterized by excess accumulation of iron in various organs and often leads to diabetes mellitus (DM). To study whether mutations in the hemochromatosis gene (HFE) could be a risk factor for the development of gestational diabetes mellitus (GDM), the prevalence of HFE mutations in patients with GDM was compared to that of healthy pregnant controls. Methods: GDM was diagnosed in 208 of 2,421 pregnant woman screened between the 24th and 28th week of gestation over a period of 18 months. Patients and 170 matched control subjects were screened for the HFE gene mutations C282Y and H63D. Results: In North and Central European GDM patients, the allele frequency of the C282Y mutation (7.7%) was higher than in pregnant controls (2.9%; p = 0.04), while the frequency of the H63D mutation was not different (p = 0.45). Three patients with GDM were homozygous for H63D (3.1%), 1 patient was homozygous for C282Y (1.0%), 2 patients were compound heterozygous (2.0%) and 26 were heterozygous [11 C282Y (11.2%) and 15 H63D (15.3%)]. C282Y and H63D allele frequencies were not different between controls and GDIVI patients of Southern European or non-European origin. Irrespective of the HIFE-mutation status, serum ferritin levels were increased in patients with GDM compared to healthy pregnant controls (p = 0.01), while transferrin saturation was similar in both groups. Conclusions: In North and Central European patients with GDM, the C282Y allele frequency is higherthan in healthy pregnant women, suggesting a genetic susceptibility to the development of GDM. Copyright (c) 2005 S. Karger AG, Basel.

Haemochromatosis and HLA--H

The recently identified can didate gene, HLA-H, for haemochromatosis (HH) by Feder et al. generated considerable scientific interest coupled with a degree of uncertainty about the likely involvement of this gene in this common iron metabolism disorder, Feder et al. found a single point mutation resulting in an amino acid substitution (C282Y) that was homozygous in 148 (83%) of their patients, heterozygous in 9 patients (5%) but completely absent in 21 patients (12%). They proposed that the lack of a causative mutation in HLA-H in 12% of their patients was because these cases were not linked to chromosome 6p. A significant weakness in this argument is that all familial studies of the disorder so far have concluded that HH is due to a single major HLA-linked gene5-7. The ultimate test for a candidate gene is the clear segregation of a mutation with the disorder in all patients. Thus, some of the uncertainty surrounding the role of HLA-H in HH may be resolved by the identification of complete concordance of the C282Y mutation (or some other mutation) in HLA H with disease status in HH families. One potential problem in the design of such an experimental analysis is that a number of studies have shown the presence of a predominant ancestral haplotype in all HH populations examined: Australian, French, Italian, UK and US Thus in the analysis of a putative causative mutation, it is important to include families with...

Novel loci affecting iron homeostasis and their effects in individuals at risk for hemochromatosis

Variation in body iron is associated with or causes diseases, including anaemia and iron overload. Here, we analyse genetic association data on biochemical markers of iron status from 11 European-population studies, with replication in eight additional cohorts (total up to 48,972 subjects). We find 11 genome-wide-significant (P<5 × 10−8) loci, some including known iron-related genes (​HFE, ​SLC40A1, ​TF, ​TFR2, ​TFRC, ​TMPRSS6) and others novel (​ABO, ​ARNTL, ​FADS2, ​NAT2, ​TEX14). SNPs at ​ARNTL, ​TF, and ​TFR2 affect iron markers in ​HFE C282Y homozygotes at risk for hemochromatosis. There is substantial overlap between our iron loci and loci affecting erythrocyte and lipid phenotypes. These results will facilitate investigation of the roles of iron in disease.

Genetic testing for haemochromatosis in patients with chondrocalcinosis

Hereditary haemochromatosis (HH) is the most common lethal monogenic human disease, affecting roughly 1 in 300 white northern Europeans. Homozygosity for the C282Y polymorphism within the HFE gene causes more than 80% of cases, with compound heterozygosity of the C282Y and H63D polymorphism also increasing susceptibility to disease. The aim of this study was to determine the frequency of the C282Y and H63D polymorphisms in the disease, and to assess the risk of HH in heterozygotes for the C282Y polymorphism. 128 patients were recruited because of either radiographic chondrocalcinosis (at least bicompartmental knee disease or joints other than the knee involved) or CPPD pseudogout. Genotyping of the HFE C282Y and H63D mutations was performed using PCR/SSP and genotypes for the C282Y polymorphism confirmed by PCR/RFLP. Historical white European control data were used for comparison. Two previously undiagnosed C282Y homozygotes (1.6%), and 16 C282Y heterozygotes (12.5%), including four (3.1%) C282Y/ H63D compound heterozygotes were identified. This represents a significant overrepresentation of C282Y homozygotes (relative risk 3.4, p-0.037), but the number of heterozygotes was not significantly increased. At a cost per test of £1 for each subject, screening all patients with chondrocalcinosis using the above ascertainment criteria costs only £64 for each case of haemochromatosis identified, clearly a highly cost effective test given the early mortality associated with untreated haemochromatosis. Routine screening for haemochromatosis in patients with appreciable chondrocatcinosis is recommended.

Prevalência da mutação do gene HFE em pacientes com porfiria cutânea tardia do Hospital Universitário Pedro Ernesto- UERJ

A Porfiria Cutânea Tardia (PCT) é uma desordem dermatológica, caracterizada por fotossensibilidade induzida pela circulação de porfirinas que se depositam na pele. Tanto a forma familial como a esporádica são desordens dependentes do acúmulo de ferro. A presença da mutação do gene da Hemocromatose (HFE) é um importante fator de risco para o acúmulo de ferro e pouco se sabe sobre sua prevalência na população brasileira. Da mesma forma, existem poucos relatos a respeito da associação entre mutação do gene HFE e Porfiria Cutânea Tardia. No presente trabalho descrevemos as frequências dos principais alelos e genótipos do gene da Hemocromatose HFE1 em uma coorte de 25 pacientes brasileiros atendidos no HUPE, com Porfiria Cutânea Tardia, durante o período de janeiro 1990 à dezembro 2012, realizando uma correlação da presença desta mutação com a sobrecarga de ferro neste grupo de pacientes. Neste estudo foi utilizado um grupo controle da população fluminense pareado por idade, sexo e grupo étnico informado, para comparar com os dados avaliados dos pacientes com PCT. A pesquisa das mutações genéticas C282Y e H63D do gene da hemocromatose ocorreu através de técnicas de PCR tempo real e e os resultados ratificados por sequenciamento de Sanger. Dos resultados encontrados, não ocorreram diferenças estatísticas significativas nas frequências alélicas e genotípicas das mutações C282Y e H63D entre a coorte com PCT e a população controle. Entretanto, há um forte indício da participação da mutação H63D em um paciente homozigoto, para desenvolvimento da doença, conforme observado na literatura. Dos ensaios bioquímicos, os níveis de ferritina encontrados entre os pacientes portadores de PCT com a mutação H63D foram maiores que os indivíduos sem a mutação.

Characterization of the degradation of wild-type and mutant HFE proteins during stress signalling in the endoplasmic reticulum

HFE is a transmembrane protein that becomes N-glycosylated during transport to the cell membrane. It acts to regulate cellular iron uptake by interacting with the Type 1 transferrin receptor and interfering with its ability to bind iron-loaded transferrin. There is also evidence that HFE regulates systemic iron levels by binding to the Type II transferrin receptor although the mechanism by which this occurs is still not well understood. Mutations to HFE that disrupt this function, or physiological conditions that decrease HFE protein levels, are associated with increased iron uptake, and its accumulation in tissues and organs. This is exemplified by the point mutation that results in conversion of cysteine residue 282 to tyrosine (C282Y), and gives rise to the majority of HFE-related hemochromatoses. The C282Y mutation prevents the formation of a disulfide bridge and disrupts the interaction with its co-chaperone β2-microglobulin. The resulting misfolded protein is retained within the endoplasmic reticulum (ER) where it activates the Unfolded Protein Response (UPR) and is subjected to proteasomal degradation. The absence of functional HFE at the cell surface leads to unregulated iron uptake and iron loading. While the E3 ubiquitin ligase involved in the degradation of HFE-C282Y has been identified, the mechanism by which it is targeted for degradation remains relatively obscure. The primary objective of this project was to further our understanding of how the iron regulatory HFE protein is targeted for degradation. Our studies suggest that the glycosylation status, and the active process of deglycosylation, are central to this process. We identified a number of additional factors that can contribute towards degradation and explored their regulation during ER stress conditions.