Genetic and metabolic factors are associated with increased hepatic iron stores in a selected population of p.Cys282Tyr heterozygotes.

Heterozygosity for p.Cys282YTyr is not ordinarily associated with a hemochromatosis phenotype, unless associated in the compound heterozygous state with other HFE mutations. The aims of the study were to identify factors responsible for iron overload in patients who were only heterozygous for p.Cys282Tyr at first genetic testing. Since 2001, twelve p.Cys282Tyr heterozygous patients with iron overload, defined by increased transferrin saturation, serum ferritin and hepatic iron stores, were identified. Four patients showed rare nonsense or missense HFE mutations in the compound heterozygous state with p.Cys282Tyr. One mutation (p.Gln233X) was never described before. The other 8 patients did not carry any other causal mutations in iron-related genes, but showed a very high prevalence of hepatic steatosis and steato-hepatitis, and metabolic alterations. Serum ferritin levels did not differ between the two groups, but transferrin saturation, hepatic iron amount and distribution significantly did. These last indices should be then strongly considered to decide for additional genetic characterization in p.Cys282Tyr heterozygotes. Our results also highlights the influence of metabolic alterations on serum iron indices and pattern of hepatic iron accumulation.

Novel mutations of the ferroportin gene (SLC40A1): analysis of 56 consecutive patients with unexplained iron overload.

The aim of this study was to search for SLC40A1 mutations in iron overloaded patients, which tested negative for HFE mutations and other iron-related genes. After a careful differential diagnosis, we selected 56 patients with unexplained iron overload whose phenotype could suggest the ferroportin disease. Iron overload was assessed by liver biopsy or by superconducting quantum interference device. SLC40A1 exons and intron-exon boundaries were amplified by polymerase chain reaction and sequenced. We also evaluated the presence of the insulin-resistance hepatic iron overload and of non-alcoholic fatty liver disease. Iron status was assessed in 44 families. We identified two novel mutations (D157N and V72F) at the heterozygous state in two probands. Phenotype heterogeneity was observed in both families, suggesting variable penetrance and expression. Including the two affected ones, 25 of the 44 families (57%) available for the iron study had one or more relatives with increased serum iron indices. Our findings not only suggest that the presence of major alterations of serum iron parameters in probands' relatives is a main criteria to improve the power of the genetic testing for ferroportin disease but also indicate that a number of patients exists in which the etiology of iron overload remains still undefined.

Iron chelation therapy in aceruloplasminaemia: study of a patient with a novel missense mutation.

We describe a novel missense mutation of ceruloplasmin in a patient with aceruloplasminaemia causing the replacement of a neutral amino acid (phenylalanine) with a polar one (serine) at position 198, probably leading to abnormal folding and secretion of the protein. The patient showed mild microcytic anaemia, mild hepatic iron overload, and marked brain iron overload. Six months of therapy with deferiprone was ineffective in removing iron from the tissues. Deferoxamine was more efficient in removing excess iron from the liver but aggravated the disease related anaemia. After more than one year of chelation treatment, the brain magnetic resonance imaging signal did not change. Overall, these findings indicate that treatment of iron overload in aceruloplasminaemia is a difficult challenge and that new iron chelators, more efficient in crossing the blood-brain barrier, are needed.

Prevalence of HFE mutations in upper Northern Italy: study of 1132 unrelated blood donors.

BACKGROUND: In the Italian general population, prevalence of C282Y is lower than in Northern European countries. We hypothesised a higher prevalence of C282Y in Northern than in Central and Southern Italy. We previously identified a nonsense mutation (W169X) in haemochromatosis probands originating from a Northern Italian region (Brianza). AIM: To define the prevalence of HFE mutations in that region. Subjects and methods. A total of 1132 unrelated blood donors from the Blood Banks of Monza and Merate were investigated for C282Y, H63D, S65C and W169X mutations by PCR-restriction assays. A total of 300 were also tested for rare HFE and TFR2 mutations by reverse-hybridization test strips. RESULTS: Two C282Y homozygotes, eight C282Y/H63D compound heterozygotes, 27 H63D homozygotes and one W169X heterozygote were found. The allele frequencies of C282Y, H63D, S65C, and W169X were 3.2, 13.4, 1.3, and 0.04%, respectively. CONCLUSIONS: Our results confirm the existence of a decreasing frequency of C282Y allele from upper to lower Northern Italy. This difference is probably related to the larger Celtic component of upper Northern Italian populations in which screening studies for haemochromatosis may even be cost effective. W169X, due to its severity, should be looked for in all haemochromatosis patients of Northern ancestry with an incomplete HFE genotype.

Two novel nonsense mutations of HFE gene in five unrelated italian patients with hemochromatosis.

BACKGROUND & AIMS: Most hemochromatosis patients of Northern European descent are homozygous for the C282Y mutation of HFE gene. In Italy, many patients with iron overload are not homozygous for C282Y, and the presence of other mutations or other genetic determinant has been suggested. METHODS: Five unrelated Italian patients heterozygous for C282Y with the classic hemochromatosis phenotype were studied. The entire coding sequence and the exon-intron boundaries of the HFE gene were analyzed. Chromosome 6p haplotypes were defined in each patient by analysis of D6S265, D6S105, and D6S1281 microsatellites. RESULTS: Two novel nonsense HFE mutations were identified in exon 3 in the C282Y negative chromosome. The first one, a G-to-T transition at codon 168, was detected in 3 probands; the second, a G-to-A transition at codon 169, was detected in the others. CONCLUSIONS: The 2 nonsense mutations in the compound heterozygous state with C282Y result in the classic hemochromatosis phenotype in several unrelated Italian patients. This confirms that hemochromatosis in Italy is not as homogeneous as in northern Europe and suggests that other mutations can exist in C282Y or H63D heterozygotes with iron overload. These findings have practical implications for diagnostic and screening strategies for hemochromatosis.

Semiquantitative and qualitative assessment of hepatic iron in patients with chronic viral hepatitis: relation with grading, staging and haemochromatosis mutations.

BACKGROUND: Hepatic iron overload is a common but still poorly characterized finding in patients with chronic viral hepatitis. AIM: To evaluate lobular and cellular distribution of iron in patients with chronic viral hepatitis, the relation between hepatic iron distribution, grading and staging, and the frequency of haemochromatosis gene mutations. PATIENTS: Thirty-four patients with chronic viral hepatitis and iron overload; 34 matched chronic viral hepatitis controls without iron overload; 139 healthy controls. METHODS: Hepatic iron was assessed by hepatic iron concentration and Deugnier's score, histological grading and staging by Ishak's score, and frequency of haemochromatosis gene mutations by polymerase chain reaction-restriction assays. RESULTS AND CONCLUSIONS: Iron deposits were found in hepatocytes (94% of the patients), sinusoidal tracts (88%) and portal cells (59%). In 41%, iron deposits were homogeneously distributed in the hepatic specimen. Hepatocytic iron showed a decreasing gradient from Rappaport's zone 1 to 3. Heavy alcohol intake influenced the distribution rather than the amount of hepatic iron by increasing sinusoidal iron. Haemochromatosis gene mutations were more frequent in chronic viral hepatitis patients with iron overload than in those without iron overload and in healthy controls suggesting they contribute to pathogenesis of hepatic iron accumulation. The correlation between hepatic fibrosis and portal iron supports the fibrogenetic role of iron in chronic viral hepatitis.