Séverine Cunat

Estrogen receptor mediated inhibition of cancer cell invasion and motility: an overview.

In this overview of results from our laboratory, we address the question of the role of estrogens during early steps of metastasis, involving cell invasion through the basement membrane and cell motility. The motility of several estrogen receptor (ER) positive breast (MCF7, T47D) and ovarian (BG-1, SKOV3, PEO4) cancer cell lines was studied using a modified Boyden chamber assay. We observed, in all cases, estradiol induced inhibition of cancer cell invasion and motility. A similar inhibitory effect of estradiol was found when the wild-type ER alpha was stably transfected in the ER-negative MDA-MB231 cells and 3Y1-Ad12 cancer cells. The mechanism of this inhibitory effect is unknown. In ovarian cancer, however, it may involve intermediary proteins such as fibulin-1, an extracellular matrix protein that strongly interacts with fibronectin and which is induced by estrogen and secreted by ovarian cancer cells. We conclude that estrogens in ER-positive breast and ovarian cancers have a dual effect, since they stimulate tumor growth but inhibit invasion and motility. This may be consistent with the good initial prognostic value of ER-positive breast cancers compared to ER negative breast cancers noted in several clinical studies.

Iron overload in HFE C282Y heterozygotes at first genetic testing: a strategy for identifying rare HFE variants

Background Heterozygotes for the p.Cys282Tyr (C282Y) mutation of the HFE gene do not usually express a hemochromatosis phenotype. Apart from the compound heterozygous state for C282Y and the widespread p.His63Asp (H63D) variant allele, other rare HFE mutations can be found in trans on chromosome 6. Design and Methods We performed molecular investigation of the genes implicated in hereditary hemochromatosis in six patients who presented with iron overload but were simple heterozygotes for the HFE C282Y mutation at first genetic testing. Functional impairment of new variants was deduced from computational methods including molecular modeling studies. Results We identified four rare HFE mutant alleles, three of which have not been previously described. One mutation is a 13-nucleotide deletion in exon 6 (c.1022_1034del13, p.His341_Ala345>LeufsX119), which is predicted to lead to an elongated and unstable protein. The second one is a substitution of the last nucleotide of exon 2 (c.340G>A, p.Glu114Lys) which modifies the relative solvent accessibility in a loop interface. The third mutation, p.Arg67Cys, also lies in exon 2 and introduces a destabilization of the secondary structure within a loop of the α1 domain. We also found the previously reported c.548T>C (p.Leu183Pro) missense mutation in exon 3. No other known iron genes were mutated. We present an algorithm at the clinical and genetic levels for identifying patients deserving further investigation. Conclusions Our results suggest that additional mutations in HFE may have a clinical impact in C282Y carriers. In conjunction with results from previously described cases we conclude that an elevated transferrin saturation level and elevated hepatic iron index should indicate the utility of searching for further HFE mutations in C282Y heterozygotes prior to other iron gene studies.

The Southern French registry of genetic hemochromatosis: a tool for determining clinical prevalence of the disorder and genotype penetrance

Background Despite great progress in understanding the mechanisms underlying genetic hemochromatosis, data on the prevalence and the penetrance of the disorder are conflicting. Design and Methods A registry of patients with genetic hemochromatosis was established in the South of France and a regional health network was developed to allow the inclusion of all the diagnosed patients. C282Y homozygous patients classified in stages 2 (biological iron overload), 3 and 4 (clinical manifestations of iron overload, stage 4 being the more severe) according to the classification of the French National Authority for Health were included in the registry over a 6-year period. Results A total of 352 symptomatic C282Y homozygotes were identified, resulting in a total prevalence of 1.83 per 10,000 (95% CI: 1.63 to 2.02) in subjects over 20 years and 2.40 per 10,000 (95% CI, 2.15 to 2.65) among subjects of European descent. Among Europeans, the total calculated penetrance was 15.8% in stage 2 or higher, 12.1% in stage 3 or 4 and 2.9% in stage 4. The penetrance was slightly higher in males (18.7%) than in females (13.2%). It was 19.9% for individuals over 40 years of age (24.1% and 16.3% in males and females, respectively) with a maximum of 31% in subjects between 50 and 54 years old. Among 249 patients with complete records, 24% were in stage 2, the majority (58%) were in stage 3, and 18% in stage 4. There was a higher proportion of males, and excessive alcohol intake was more prevalent in stage 4 than in stages 2 and 3 combined. Conclusions A French Mediterranean regional hemochromatosis registry with strict inclusion criteria is a useful tool for characterizing the history of this disease, particularly for the most severely affected patients, as defined by the disease severity classification. The total prevalence of symptomatic C282Y homozygotes in the region was found to be low. However, clinical penetrance (stages 3 and 4) was not negligible.

Variable age of onset and clinical severity in transferrin receptor 2 related haemochromatosis: novel observations.

Since the discovery of the HFE gene and the C282Y mutation (Type 1 haemochromatosis), new genes involved in iron metabolism have been described. Juvenile haemochromatosis has been related to HFE2 and HAMP mutations (Type 2A and 2B) and is described as severe iron overload affecting patients before the age of 30 years (Brissot et al, 2011). Mutations in the TFR2 gene lead to Type 3 haemochromatosis whose clinical picture mimics Type 1. However, rare cases affecting young patients have been reported (Brissot et al, 2011). The ferroportin disease has been linked to SLC40A1 mutation and is described as iron overload affecting patients at any age (Le Lan et al, 2011). The basic mechanism accounting for iron overload in types 1, 2 and 3 haemochromatosis is decreased hepcidin synthesis. TFR2 is also involved in hepcidin synthesis regulation (Wallace et al, 2005; Gao et al, 2010) but its definite mode of action remains to be determined. In Type 1 haemochromatosis, cofactors play an important role because clinical penetrance of C282Y homozygosity is low (Allen et al, 2008). In Type 3 haemochromatosis, clinical expression seems high but reported cases are too scarce to definitely assess penetrance. Here, we report seven new cases of Type 3 haemochromatosis. Transferrin receptor 2 mutations were screened as part of the diagnostic activity of the French Reference Centre for Rare Iron Overload Diseases of Genetic Origin and the associated network of expertise Centres. Written informed consent was obtained and the study was performed in accordance with the Declaration of Helsinki and with the French regulations on medical genetic diagnosis. Patients were tested for HFE mutations (p.Cys282Tyr, p.His63Asp) (Jouanolle et al, 1996; Aguilar Martinez et al, 1997). The entire coding region and intronic flanking sequences of the TFR2 gene (NCBI NM_003227.3, NP_003218.2) were sequenced. To exclude other mutation(s), analyses of the haemochromatosis type 2 (juvenile) (HFE2), hepcidin (HAMP), and ferroportin (SLC40A1) coding sequences were performed. To determine the potential consequences of mutations on the protein, TFR2 amino acid sequence and mutations were input as required into the following algorithms: Scale-Invariant Feature Transform (SIFT; http://sift.jcvi.org/), Polymorphism Phenotyping v2 (Polyphen-2; http://genetics.bwh. harvard.edu/pph2/bgi.shtml), point mutant (Pmut; http:// mmb2.pcb.ub.es:8080/PMut/), SNPs3D (http://www.snps3d. org/), and Scalable Nucleotide Alignment Program (SNAP; http://www.rostlab.org/services/snap/). Seven unrelated patients were diagnosed with Type 3 haemochromatosis. Three were homozygous for the previously undescribed p.Asn412Ile (c.1235A>T), p.Gly430Arg (c.1288G>A), p.Arg678Pro (c.2033G>C) TFR2mutations. Consanguinity was likely only for Patient 1. Four patients were compound heterozygotes for at least one new TFR2 mutation each: p.Leu85_A a96delinsPro (c.254_286 + 9del), p.Met705Hisfs*87 (c.211 2dup), p.Arg730Cys (c.2188C>T), p.Gly735Ser (c.220 3G>A), p.Trp781* (c.2343G>A). One patient, who was a compound heterozygote for two previously described TFR2 mutations (p.Ala444Thr, p.Gly792Arg) (Lee & Barton, 2006; Biasiotto et al, 2008) was found to carry the p.Gly204Ser mutation in the SLC40A1 gene coding for ferroportin. The main clinical and biological features of the patients are summarized in Table I. No mutations were found in the HAMP and HFE2 genes of the patients. Sequencing of the HFE gene revealed no other mutations than the H63D or C282Y (Table I). Patients 1 and 2 were diagnosed earlier than usually described for Type 3 haemochromatosis. Patient 1 was referred for major asthenia at the age of 10 years. Biological workup found high transferrin saturation leading to the diagnosis of haemochromatosis; an echocardiogram revealed no abnormalities. Patient 2 was diagnosed as a result of elevated transferrin saturation in the context of a1-antitrypsin deficiency. Patients 3, 4 and 5 were diagnosed between 20 and 30 years of age. Patient 3 originated from North Africa and was diagnosed upon arrival in France, which could explain the older age at diagnosis. The daughter of Patient 3 was heterozygous for the TFR2:p.Gly430Arg mutation and had normal iron parameters. The parents of Patient 4 were heterozygous for mutation p.Gly735Ser and p.Leu85_Ala96delinsPro respectively, and had normal iron parameters. Patient 6 was diagnosed with non-HFE related haemochromatosis at the age of 28 years. Diagnosis of ferroportin disease was made later by finding the SLC40A1:p.Gly204Ser mutation. The clinical presentation was unusual with elevated transferrin saturation and arthropathy. Moreover, phlebotomies were very well tolerated and removed 19 5 g iron. For these reasons, sequencing of other genes related to iron metabolism was performed and revealed two already described mutations in TFR2. Patient 7 was diagnosed during the diagnostic workup of post-partum infection. She had elevated liver enzymes, and transient elastography (Fibroscan , Echosens, Paris, France)

HAMP promoter mutation nc.-153C>T in non p.C282Y homozygous patients with iron overload

In a recent paper, Island and colleagues[1][1] described a heterozygous hepcidin ( HAMP ) promoter mutation, nc.- 153C>T, which in association with HFE p.C282Y homozygosity appeared to lead to very severe iron overload (IO). They demonstrated in vitro that this HAMP mutation decreases

The role of genetic factors in patients with hepatocellular carcinoma and iron overload - a prospective series of 234 patients.

Iron overload (IO) in HFE‐related hereditary haemochromatosis is associated with increased risk of liver cancer. This study aimed to investigate the role of other genes involved in hereditary IO among patients with hepatocellular carcinoma (HCC).

Homozygous mutation of the 5'UTR region of the L-Ferritin gene in the hereditary hyperferritinemia cataract syndrome and its impact on the phenotype

Hereditary hyperferritinemia cataract syndrome (HHCS) is an autosomal dominant disorder characterized by hyperferritinemia without iron overload and early-onset bilateral cataract induced by L-ferritin accumulation in the lens.[1][1] Affected patients harbor heterozygous mutations within the iron

Absence of functional mutations in the ferroportin-encoding SLC40A1 gene in porphyria cutanea tarda: a series of 37 cases from southern France.

The presence of iron overload in porphyria cutanea tarda (PCT) is well-known, although the overload is not always concentrated in the liver as was originally thought. The origin of this overload is not known with certainty in all cases and is probably explained by various, most often genetic, mechanisms. From this perspective, the implication of protein activity-modifying mutations or polymorphisms in key genes involved in iron metabolism regulation has already been well documented, with deleterious HFE mutations being the most frequently identified changes, occurring in up to one-third of patients (1). Conversely, other genes involved in the regulation of iron metabolism (haemojuvelin, hepcidin, type 2 transferrin receptor) have also been analysed in previous reports with negative results. Ferroportin is another important player in iron metabolism. We report here the search for functionally relevant changes in the ferroportin-encoding SLC40A1 gene in a series of patients with PCT, the first analysis of its kind. Thirty-seven consecutive patients diagnosed with PCT based on the usual clinical and biochemical criteria (25 men and 12 women, age range 34–71 years, including 5 patients with familial-type disease), all originating from southern France, were studied. After receiving written consent, genomic DNA was extract ed from peripheral blood mononuclear cells and subjected to SLC40A1 analysis using stringent amplification con ditions. Then, its eight exons and flanking areas were directly sequenced. The HFE gene was also analysed for functionally significant mutations (homozygous or heterozygous C282Y mutations and the homozygous H63D mutation). (The heterozygous H63D mutation was not studied since its possible effect on iron metabolism remain doubtful and it is frequently encountered in the Mediterranean basin.) Analysis of the HFE and SLC40A1 genes was performed in all 37 patients. In the case of SCL40A1, a number of known polymorphisms with no influence on the primary structure of the encoded protein were identified. A previously unreported complex genomic

Inherited or acquired modifiers of iron status may dramatically affect the phenotype in dehydrated hereditary stomatocytosis

Severe iron overload is frequent in dehydrated hereditary stomatocytosis (DHSt) despite well‐compensated hemolysis and no or little transfusion requirement. We investigated 4 patients with proven DHSt, in whom the degree of hemolysis was closely related to iron status. Genetic modifiers increasing iron stores (HFE:pCys282Tyr, HAMP:c‐153C>T mutations) were accompanied with high liver iron concentrations and increased hemolysis, whereas therapeutic phlebotomies alleviated the hemolytic phenotype. There were no manifestations of hemolysis in one patient with low iron stores. Hemolysis reappeared when iron supplementation was given. The search for genetic or acquired modifiers of iron status and the modulation of iron stores may help in the management of these patients.