Genotype–phenotype correlation in a Spanish population homozygous for the H63D mutation of the HFE gene

Abstract

Hereditary hemochromatosis (HH) is an autosomal recessive disorder of iron metabolism with excessive cellular iron levels resulting in tissue damage [1–3]. In 1996, Feder et al. showed that in most HH patients, the HFE gene is mutated in position 282 (replacement of cysteine by tyrosine, C282Y) [4]. In Spain, 85–90% of HH patients showed this mutation [5, 6]. However, a second mutation, the replacement of histidine in position 63 by aspartic acid (H63D), is much more frequent. In our area, the Northeast of Spain, the H63D mutation was present in 37% of the general population, and 4% were homozygous [7]. In a murine model, the contribution of the H63D homozygosity (DD genotype) was demonstrated. However, the iron overload (IO) was below that of the C282Y homozygosity (YY genotype) [8]. Moreover, data in humans support that the DD genotype could result in IO [9–11]. Given the high prevalence of this mutation in our population, we evaluated the genotype–phenotype correlation in homozygotes for the H63D mutation. Two studies were conducted in two Spanish populations. First, the correlation between the HFE mutations and the amount of iron in the liver was evaluated in 78 cases (7 cases were DD homozygotes) [12]. Liver biopsy was carried out because of a biochemical IO [serum transferrin saturation >50% and/or serum ferritin >350 μg/l]. Hemochromatosis was considered when the hepatic iron index (HII) exceeded 1.9 μmol/g of dry weight multiplied by the age in years, μmol/g.year [1–3]. In 30 out of 78 cases, HII exceeded 1.9 μmol/g.year, including three cases (10%) homozygous for the H63D mutation (HII=4.2, 3.9, and 3.8 μmol/g.year). Moreover, in one patient homozygous for the H63D mutation, the HII was 1.8 μmol/g.year Therefore, four out of the seven DD homozygotes showed moderate liver IOs (57%). C282Y homozygotes showed the highest levels of HII. DD homozygotes had higher levels of HII than wild-type patients and H63D heterozygotes (Table 1). The second study was conducted between 1998 and 2004 in 55 consecutive patients with the DD genotype. All patients came from our university hospital and were studied for all the known secondary causes of biochemical IO [13, 14]. Data were not available in one case, which was excluded from the study. Hepcidin (HAMP) gene mutations were investigated given that these mutations could modify the severity of the IO [15]. Transferrin saturation and serum ferritin were evaluated by automated methodologies (Hitachi 911 and Elecsys, Roche). Moreover, total iron removed by phlebotomy was calculated in grams. Mutations of the HFE gene (C282Y, H63D, and S65C) were analyzed with LightCycler equipment [6, 7, 14]. The HAMP gene was evaluated using single strand conformational polymorphism and direct sequencing [15]. The patients were 36 men and 18 women, with a median age was 51 years. They were chosen for the study because of a biochemical IO (41 cases) or a family study (13 cases). Forty-five patients (83%) showed a biochemical IO pattern. An underlying cause of biochemical IO was observed in 35 A. F. Remacha (*) . M. P. Sarda . M. J. Barcelo . V. Bach . A. Altes . M. Baiget . C. Guarner . I. Blesa Hematology Department, Hospital de Sant Pau, Avda Padre Claret 167, Barcelona, 0825, Spain e-mail: aremacha@hsp.santpau.es