Jean-Yves Le Gall

Insulin resistance–associated hepatic iron overload

BACKGROUND & AIMS Hepatic iron overload has been reported in various metabolic conditions, including the insulin-resistance syndrome (IRS) and nonalcoholic steatohepatitis (NASH). The aim of this study was to show that such hepatic iron overload is part of a unique and unrecognized entity. METHODS A total of 161 non-C282Y-homozygous patients with unexplained hepatic iron overload were included. We determined the age; sex; presence of IRS (1 or more of the following: body mass index of >25, diabetes, or hyperlipidemia); serum iron tests and liver iron concentration (LIC; reference value, <36 micromol/g); liver function test results; C282Y and H63D HFE mutations; and liver histological status. RESULTS Patients were predominantly male and middle-aged. Most (94%) had IRS. Transferrin saturation was increased in 35% (median, 42%; range, 13%-94%). LIC ranged from 38 to 332 micromol/g (median, 90 micromol/g), and LIC/age ratio ranged from 0.5 to 4.8 (median, 1.8). Allelic frequencies of both HFE mutations were significantly increased compared with values in normal controls (C282Y, 20% vs. 9%; H63D, 30% vs. 17%), only because of a higher prevalence of compound heterozygotes. Patients with no HFE mutations had similar degrees of iron overload as those with other genotypes, except for compound heterozygotes, who had slightly more iron burden. Steatosis was present in 25% of patients and NASH in 27%. Portal fibrosis (grades 0-3) was present in 62% of patients (grade 2 or 3 in 12%) in association with steatosis, inflammation, and increased age. Sex ratio, IRS, transferrin saturation, and LIC did not vary with liver damage. Serum ferritin concentration, liver function test results, and fibrosis grade were more elevated in patients with steatosis and NASH than in others, but LIC and allelic frequencies of HFE mutations were similar. CONCLUSIONS This study shows that patients with unexplained hepatic iron overload are characterized by a mild to moderate iron burden and the nearly constant association of an IRS irrespective of liver damage.

Phenotypic expression of HFE mutations: A French study of 1110 unrelated iron-overloaded patients and relatives ☆ ☆☆

BACKGROUND & AIMS Two mutations have been described in the HFE gene: C282Y and H63D. The aim of this study was to determine the phenotype of the different HFE genotypes. METHODS Clinical symptoms and iron data were examined according to HFE genotypes in 531 unrelated patients with unexplained liver iron overload and 579 relatives of hemochromatotic patients. RESULTS Non-C282Y +/+ patients did not markedly differ in terms of iron overload or clinical expression according to genotype, except for compound heterozygotes, who had slightly increased transferrin saturation. This contrasted with the strikingly increased expression in C282Y homozygotes. Similar phenotype/genotype correlations were observed in relatives based on serum iron test results. Family transmission of iron overload linked to HFE was exceptional in non-C282Y +/+ siblings and frequent in C282Y homozygotes. CONCLUSIONS Iron overload in patients with the non-C282Y +/+ genotype is mild to moderate, strikingly lower than in C282Y homozygotes, and is not influenced by HFE genotype, except, to a small extent, for compound heterozygotes. The role of H63D mutation therefore seems to be marginal.

A genotypic study of 217 unrelated probands diagnosed as "genetic hemochromatosis" on "classical" phenotypic criteria.

BACKGROUND/AIMS The HFE gene is a crucial candidate gene for hemochromatosis. The aims of this study were to assess the HFE genotypic profile in a large series of unrelated probands diagnosed as having phenotypic hemochromatosis, to characterize the sub-group of patients who were not homozygous for the major C282Y mutation, and to report the iron status of the detected HFE-identical siblings. METHODS In 217 patients, the phenotypic diagnosis of hemochromatosis was based on strict bioclinical and/or histological criteria, and their genotypic profile (C282Y and H63D mutations) was determined. RESULTS 1) 209 of the 217 probands were C282Y +/+. In 33 cases, an HFE-identical sibling was identified. Two of them had neither a clinical nor a biochemical phenotypic profile of hemochromatosis in the absence of any external factor which might have attenuated this expression. 2) Eight patients (seven males) were not C282Y +/+. Their genotypic profiles were: (C282Y +/-): six cases (four were H63D +/- and two H63D -/-); (C282Y -/-): two cases (one was H63D +/+, one H63D +/-). Phenotypic expression consisted of six cases of mild liver siderosis (among whom were the four compound heterozygotes and one case of alcoholic cirrhosis) and two severe cases of hepatic iron overload (one with alcoholic cirrhosis). Three HFE-identical siblings were identified, none of them presenting with iron excess. CONCLUSIONS In our population: 1) The classical phenotypic criteria fitted, in 96.3% of cases, with a homogeneous genotypic entity defined by homozygosity for the C282Y mutation. Incomplete penetrance of the homozygous status was shown by the absence of the hemochromatosis phenotypic profile in 6% of the HFE-identical siblings. 2) A minority (3.7%) were not homozygous for C282Y. These were essentially men with mild iron overload, and might present with distinct iron overload entity(ies) as suggested by the presence in three of an HFE-identical sibling with absence of iron overload.

Novel mutation in ferroportin 1 gene is associated with autosomal dominant iron overload

We report a family affected with dominant autosomal iron overload related to a new mutation in ferroportin 1, a transmembrane protein involved in the export of iron from duodenal enterocytes and likely from macrophages. The originality of this family is represented by the nature of the mutation consisting in the replacement of glycine 490 with aspartate. Clinicians should be aware of this novel iron overload entity, which corresponds to a particular phenotypic expression (high serum ferritin values contrasting with relatively low transferring saturation, and important Kupffer cell iron deposition as compared to hepatocytic iron excess) with poor tolerance of venesection therapy and a dominant pattern of inheritance. Given this dominant transmission, the mixed Causasian-Asian origin of our Asian proband leaves open the issue of the ethnic origin of the new mutation.

Gender‐specific phenotypic expression and screening strategies in C282Y‐linked haemochromatosis: a study of 9396 French people

Summary. Most features of C282Y‐linked haemochromatosis support the implementation of population screening of the disorder in Caucasians. However, the penetrance of C282Y homozygosity is poorly documented and the strategy for population screening remains debated. Nine thousand three hundred and ninety‐six subjects (3367 men, aged 25–40 years, and 6029 women, aged 35–50 years), attending three Health Appraisal Centres, were genotyped and assessed with respect to clinical and biochemical signs of haemochromatosis. Discriminant, logistic regression and graphic analysis were used to predict homozygosity. Results were validated in 135 homozygotes detected through other family and population studies. Fifty‐four subjects (10 men and 44 women) were homozygous for C282Y. All men had abnormal iron status and most had mild clinical symptoms compatible with haemochromatosis. Identification of all homozygous men required a transferrin saturation (TS) threshold of 50% in the study group (90% specificity) and of 40% in the validation group. Homozygous women differed clinically from non‐homozygotes for the presence of distal arthralgias only (18%vs 6%, P < 0·03). Thirteen (29%) were iron‐deficient (serum ferritin < 13 µg/l) and undetectable by biochemical tests. Although the population studied was not fully representative of the general population, our data strongly suggests that, in young men, large‐scale screening for C282Y homozygosity is justified and can be achieved by using TS prescreening. However, in premenopausal women, large‐scale screening remains to be justified with respect to the natural history of haemochromatosis and should be directly genotypic.

Five distinct biological processes and 14 differentially expressed genes characterize TEL/AML1 -positive leukemia

BackgroundThe t(12;21)(p13;q22) translocation is found in 20 to 25% of cases of childhood B-lineage acute lymphoblastic leukemia (B-ALL). This rearrangement results in the fusion of ETV6 (TEL) and RUNX1 (AML1) genes and defines a relatively uniform category, although only some patients suffer very late relapse. TEL/AML1-positive patients are thus an interesting subgroup to study, and such studies should elucidate the biological processes underlying TEL/AML1 pathogenesis. We report an analysis of gene expression in 60 children with B-lineage ALL using Agilent whole genome oligo-chips (44K-G4112A) and/or real time RT-PCR.ResultsWe compared the leukemia cell gene expression profiles of 16 TEL/AML1-positive ALL patients to those of 44 TEL/AML1-negative patients, whose blast cells did not contain any additional recurrent translocation. Microarray analyses of 26 samples allowed the identification of genes differentially expressed between the TEL/AML1-positive and negative ALL groups. Gene enrichment analysis defined five enriched GO categories: cell differentiation, cell proliferation, apoptosis, cell motility and response to wounding, associated with 14 genes -RUNX1, TCFL5, TNFRSF7, CBFA2T3, CD9, SCARB1, TP53INP1, ACVR1C, PIK3C3, EGFL7, SEMA6A, CTGF, LSP1, TFPI – highlighting the biology of the TEL/AML1 sub-group. These results were first confirmed by the analysis of an additional microarray data-set (7 patient samples) and second by real-time RT-PCR quantification and clustering using an independent set (27 patient samples). Over-expression of RUNX1 (AML1) was further investigated and in one third of the patients correlated with cytogenetic findings.ConclusionGene expression analyses of leukemia cells from 60 children with TEL/AML1-positive and -negative B-lineage ALL led to the identification of five biological processes, associated with 14 validated genes characterizing and highlighting the biology of the TEL/AML1-positive ALL sub-group.

A continuous restriction map from HLA-E to HLA-F. Structural comparison between different HLA-A haplotypes

The class I region of the human major histocompatibility complex contains genes encoding the classical transplantation antigens (HLA-A, B, and C), at least three new class I genes (HLA-E, F, and G) and many class I pseudogenes (including HLA-H). By pulse field gel electrophoresis and using five rare cutter enzymes, we have constructed a precise and continuous map of 1200 kilobases (kb) around HLA-A. The blots were hybridized with HLA-A, E, and F-specific probes and with new probes derived from yeast artificial chromosomes and cosmids of the class I region. We have compared the genomic organization of the same 1200 kb in three homozygous lymphoblastoid cell lines corresponding to three different HLA haplotypes (A3, A24, and A31). The differences in size observed may have been caused by insertions and deletions and may prove valuable in understanding the evolution of the HLA chromosomal region.

Anonymous markers located on chromosome 6 in the HLA-A class I region: allelic distribution in genetic haemochromatosis

SummaryTwo yeast artificial chromosomes of the HLA class I region were subcloned. Four of the subclones studied displayed restriction polymorphisms that corresponded to six bi-allelic series. Allelic distribution of the anonymous markers was then studied by comparing a control population with a group of patients with familial haemochromatosis. Only one marker presents an unequivocal association with the haemochromatosis gene and is 100kb centromeric to HLA-A. This association however is not as strong as with HLA-A3. The results suggest two possible locations for the haemochromatosis gene: less than 100kb centromeric to the HLA-A locus, or on the telomeric side.

Evaluation of ETF1/eRF1, mapping to 5q31, as a candidate myeloid tumor suppressor gene

Interstitial deletion of the long arm of chromosome 5 is a recurrent abnormality, mainly associated with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), and it has been proposed therefore that the deleted region may contain a myeloid tumor suppressor gene. We have recently mapped a human translation termination factor gene, ETF1, to band 5q31 at D5S500, and thus to the smallest commonly deleted segment. We have evaluated ETF1 as a candidate myeloid tumor suppressor gene by analysis of the human acute myeloid leukemia cell line HL60, and of patients suffering from malignant myeloid diseases with cytogenetically-defined abnormalities of chromosome 5. Fluorescence in situ hybridization analysis revealed hemizygous loss of the ETF1 locus in HL60 cells and in four of five leukemic samples, but no inactivating mutations were identified by sequencing of the remaining ETF1 allele.

Human release factor eRF1: structural organisation of the unique functional gene on chromosome 5 and of the three processed pseudogenes

In lower and higher eukaryotes, a family of tightly related proteins designated eRF1 (for eukaryotic release factor 1) catalyses termination of protein synthesis at all three stop codons. The human genome contains four eRF1 homologous sequences localised on chromosomes 5, 6, 7 and X. We report here the cloning and the structural analysis of the human eRF1 gene family. It appears that the gene located on chromosome 5 alone is potentially functional, whereas the other three sequences resemble processed pseudogenes. This is the first description of the structural organisation of the human eRF1 gene, which has been remarkably conserved during evolution and which is essential in the translation termination process.