ALBERT

Description: Introduction: An insufficient production of hepcidin, the master regulator of iron metabolism, is recognized as the key pathogenetic feature of HFE-related hereditary hemochromatosis (HH). There is a growing interest in measuring the hepcidin levels, which may improve diagnosis, prognostic evaluation and clinical management of HH. Nevertheless, few investigative tools are available: an immunodot method for urinary hepcidin developed by a single centre (UCLA), not yet ready for large-scale diffusion, and mass spectrometry (MS) based assays, such as surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF-MS). The latter is well suited to small peptides like hepcidin, and can rapidly analyze crude samples with high throughput. Until now, urinary hepcidin has been measured by SELDI-TOF-MS only in small groups of C282Y homozygous patients, the majority of them under phlebotomy treatment. No data are available on C282Y/H63D compound heterozygotes, that can develop a milder clinical form of HH. This study was aimed to measure urinary hepcidin levels by SELDI-TOF-MS in a large group of HH patients. Methods: We used a protocol based on PBSIIc mass spectromer and Normal Phase chips similar to that recently proven successful for semi-quantitative detection of urinary hepcidin. Urinary samples from 30 control subjects were compared to those obtained from 80 HH patients (57 C282Y homozygotes, 23 C282Y/H63D compound heterozygotes). Eighteen C282Y homozygotes and 11 C282Y/H63D compound heterozygotes were analyzed at diagnosis, the remainder during maintenance phlebotomy (at least 30 days from last phlebotomy). Results: C282Y homozygotes had significantly lower urinary hepcidin levels vs. controls either at diagnosis, or after phlebotomy (P 〈 0.05). C282Y/H63D compound heterozygotes had hepcidin levels at diagnosis similar to controls, while the hepcidin:ferritin ratio was significantly decreased (P 〈 0.001) suggesting a relatively inappropriate hepcidin production. Moreover, also in this group means hepcidin levels after phlebotomy were significantly lower than in controls (P 〈 0.001). Samples from 12 randomly selected control subjects were sent to UCLA for duplicate measurement by the immunodot method, yielding a good correlation (r= 0.77; P

Description: Inadequate hepcidin synthesis leads to iron overload in HFE-related hemochromatosis. We explored the regulation of hepcidin by iron in 88 hemochromatosis patients (61 C282Y/C282Y, 27 C282Y/H63D) and 23 healthy controls by analyzing urinary hepcidin before and 24 hours after a 65-mg oral iron dose. Thirty-four patients were studied at diagnosis and had iron overload, and 54 patients were iron depleted. At diagnosis, hepcidin values in C282Y homozygotes were similar to controls, whereas values in C282Y/H63D heterozygotes were higher (P = .02). However, the hepcidin/ferritin ratio was decreased in both homozygotes (P 〈 .001) and heterozygotes (P = .017), confirming the inadequate hepcidin production for the iron load with both genotypes. In iron-depleted patients of both genotypes studied at a time remote from phlebotomy, basal hepcidin was still lower than in controls (P 〈 .001 for C282Y/C282Y and P = .002 for heterozygotes). After an iron challenge, mean urinary hepcidin excretion increased in controls (P = .001) but not patients, irrespective of genotype and iron status. Significant hepcidin increase ( ≥ 10 ng/mg creatinine) was observed in 74% of controls, 15% of homozygotes, and 32% of heterozygotes. The hepcidin response to oral iron is blunted in HFE-related hemochromatosis and not improved after iron depletion. The findings support the involvement of HFE in iron sensing and subsequent regulation of hepcidin.

Description: Background: Mutations in the iron exporter Ferroportin (Fpn) lead to type IV hemochromatosis (Ferroportin Disease, FD), a dominantly inherited disorder with heterogeneous clinical and biochemical patterns. Some patients present with predominant macrophage iron overload (M), marked elevation of serum ferritin, normal-to-low transferrin saturation (TS), and, possibly, iron restricted erythropoiesis. Others present with a phenotype resembling classical HFE-related hemochromatosis, i.e. characterized by high TS and predominant hepatocyte iron overload (H). These differences are thought to reflect heterogeneity in the functional behaviour of Fpn mutant proteins. Methods: Two unrelated probands referring to the Centre for Iron Overload Disorders in Verona because of non-HFE hemochromatosis were screened for Fpn mutations by DHPLC (Cremonesi L, Br J Haematol 2005). The functional behaviour of mutants Fpn was studied by generating Fpn-GFP constructs transfected into different cell types (HEK293T, Cos7, and mouse bone marrow macrophages), and analyzing their cellular localization, as well as their capabilities to bind hepcidin and export iron (De Domenico I, PNAS 2005). The two mutations were also expressed in zebrafish, to evaluate their impact on iron-dependent erythropoiesis. Results: Patient 1, a 59 year old male, had clinical, biochemical (TS 74.8%, ferritin 9,000 μg/l), and pathological features (marked iron overload in either macrophages and hepatocytes, absence of overt cirrhosis) somewhat ambiguous, possibly suggesting a type M Fpn variant with late secondary hepatocyte overload. He was found to be heterozygous for the new L233P mutation. Functional studies revealed that Fpn L233P does not appropriately traffic to the cell surface, resulting in inappropriate inhibition by hepcidin. Fpn L233P expression in vivo in zebrafish resulted in iron limited erythropoiesis, consistent with a type M mutation leading to macrophage iron retention. Patient 2, a 59 year old female, had features more clearly suggesting a type M Fpn variant (TS 22.7%, ferritin 1,771 μg/l, macrophage iron load), but tolerated very well phlebotomies without developing signs of anemia. She was found to be heterozygous for the new I152F mutation. Functional studies revealed a unique pattern (never observed until now), since Fpn I152F localized appropriately on cell membrane, bound near normally to hepcidin, but showed a “primary” deficit of iron export capability. I152F expression in zebrafish resulted in a trend towards iron limited erythropoiesis, though quantitatively less clear than L223P. Conclusions: FD is a heterogeneous disease caused by generally “private” mutations in Fpn. The clinical, biochemical, and pathological features vary depending on the different behaviour of mutant Fpn. In vitro and in vivo molecular expression studies are very useful to clarify the pathophysiogical spectrum of this disease.

Description: Background: Hereditary Hemochromatosis, once considered a monogenic disorder, is now seen as a polygenic disease, with clinical phenotype also influenced by several environmental factors. Besides the classic HFE gene, other genes involved in modulation of iron homeostasis and clinical phenotype include those coding for hemojuvelin and hepcidin (both responsible for Juvenile Hemochromatosis), ferroportin, and, possibly, H-ferritin. Methods: we used DHPLC to scan mutations in the above mentioned genes in 55 consecutive patients recently referred to our tertiary care unit for iron overload disorders. Many of them had at least biochemical signs of iron overload not explained, or not completely explained, by classic or rare HFE mutations. Main results: the −72 C→T variation in the promoter of hepcidin gene, near the putative TATA box, was found in a H63D heterozygous male with unexplained biochemical signs (serum ferritin 660 μg/L, TS 45%), who, rather, should have been protected by several previous blood donations. We recently found this new hepcidin mutation in a family from another cohort (Biasiotto et al., in press): genotype/phenotype correlation data were also consisting with a functional role of this mutation. Two other hepcidin variations were found in this series, the 108 G→A (new), and the 212 G→A. While the first appeared silent, familial study suggests that the second may be functional in association with C282Y. No new or functional variations were found in hemojuvelin or H-ferritin genes. Several polymorphisms were detected in the ferroportin gene, including two new sequence variations (−116 T→C in the promoter; 691+20C C→T in intron 4), that occurred in two unrelated subjects with wild-type HFE genotype. Their functional role is currently under investigation by extensive family studies and/or quantitative evaluation of hepatic iron by MRI/liver biopsy. Conclusions: DHPLC scan of iron genes appears as a helpful tool for integrating clinical data in selected patients referring for suspected iron overload, as well as for rapid identification of new mutations.

Description: Background: Hyperhomocysteinemia (HHCY), a known risk factor for thrombosis, is frequently found in patients with chronic ischemic heart disease, but its independent prognostic impact is still controversial. Aim: To evaluate the relationship between HHCY and mortality in patients after coronary artery bypass grafting (CABG) surgery. Methods: We prospectively followed 353 patients (mean age 60.1 ± 9.0; 83.3% males) who underwent elective CABG between May 1996 and May 1999. Results: after a median follow-up of 58 months, 36 patients (10.1%) had died, 28 because of cardiovascular events (Figure 1). Baseline homocysteine levels 〉90th of distribution (i.e. 〉25.2 μmol/L) were independently associated to total mortality: OR = 2.39, 95% CIs 1.02–5.59, P=0.035, after adjustment for age, sex, baseline renal function (glomerular filtration rate estimated by MDRD equation), and high-sensitivity C Reactive Protein, by logistic regression. HHCY was also independently associated with mortality due to cardiovascular events, adjusted as above including the presence or absence of myocardial infarction prior to CABG (OR = 2.66, 95% CIs 1.02–6.96, P=0.045). Conclusions: Moderate (〉25.2 μmol/L) HHCY is an independent predictor of total and cardiovascular mortality after CABG. At least in this specific subgroup of cardiovascular patients, low-cost strategies to lower homocysteine (i.e. supplementation with folate and B vitamins) should be implemented. Figure Figure