Early-onset haemochromatosis caused by a novel combination of TFR2 mutations(p.R396X/c.1538-2 A>G) in a woman of Italian descent

Haemochromatosis (HC) refers to a group of inherited disorders of iron metabolism characterized by progressive iron accumulation in parenchymal cells. If not recognized and treated, iron loading impairs the function of target organs and damages their structure. HC includes the historical and predominant HFE-related condition, and rarer conditions which have more recently been associated with mutations in the hemojuvelin (HJV), hepcidin (HAMP) and transferrin receptor 2 (TFR2) genes. HC also involves certain mutations of the ferroportin gene (SLC40A1), albeit most of the SLC40A1-related patients display a distinct iron overload syndrome that is recognized as the ferroportin disease.1

In the present study, we aimed to identify the genetic basis of iron overload in a 28-year-old woman of Italian descent. The patient was referred to us, in the context of a premarital medical evaluation, because of high levels of serum iron (381 μg/dL; Normal range: 50–160), transferrin saturation (determined twice: at 80 and 93%, respectively) and serum ferritin (900 μg/L; N: 10-291). She did not present clinical manifestations of HC, except of astheny. Secondary causes of iron overload were excluded. The patient denied any history of alcohol abuse or blood transfusion, serologies for hepatitis A and C were negative, vaccination against hepatitis B had been performed, and hematological constants were in the normal range (hemoglobin: 13 g/dL, white blood cells: 7.15 10/L, platelets: 355x10/L). In addition, the patient had normal levels of liver enzymes (ASAT, ALAT, γ-GT) and normal blood sugar parameters, while moderate increases in C reactive protein concentration (8 mg/L, N<5) and Erythrocyte Sedimentation Rate (36, N<15) were observed. Because of the clearly elevated serum ferritin concentration, the hepatic iron overload was investigated by magnetic resonance imaging (MRI). It was estimated to be quite high (with a Hepatic Iron Content of 310±50 μg/L, N <36; the calculation being performed with the algorithm developed by Gandon Y. and collaborators,2 for a magnetic field of 1.5 Tesla). Imaging further showed that iron deposits were homogeneously distributed. No liver biopsy was performed. At present, the patient is included in a phlebotomy program. Although 2.3g of iron had been removed, both the serum ferritin concentration (279 μg/L) and the transferrin saturation level (53%) remain elevated. Of note, the C reactive protein concentration had been normalized (1 mg/L).

After obtaining written informed consent, we searched for the p.C282Y, p.H63D and p.S65C HFE variations. As this specific screening was negative, we scanned the entire coding regions and splicing junctions of the five haemochromatosis genes. This allowed us to only detect two mutations in the TFR2 gene (Genbank # NM_003227.3): a heterozygous C>T transition at nucleotide c.1186 in exon 9 (Figure 1A), and a heterozygous A>G transition at nucleotide c.1538-2, in intron 12 (Figure 1B). The c.1186 C>T substitution results in a premature stop codon at amino-acid 396 (p.R396X). This mutation is not new, since it has been previously reported in an haemochromatosis patient of Scottish descent.3 The novel c.1538-2 A>G substitution abrogates the intron 12 acceptor splicing site as it changes the critical AG sequence into GG. This splicing mutation is expected to dramatically modify the TFR2 reading frame, which encompasses 18 exons and ends with nucleotide triplets encodings the TFR2 dimerization domain. This was not experimentally verified because of the paucity of TFR2 mRNA in circulating blood cells.

Figure 1.Sequencing electrophoregrams showing the mutated TFR2 exon 9 (A) and intron 12 (B) forward heterozygous sequences (position of the c.1186 A>G and c.1538-2 A>G nucleotide change are indicated by arrows and the intron 12/exon 13 junction is delimited by dashes).

It is well recognized that iron overload phenotypes observed in HC are highly variable. Taking into account the influence of environmental factors and of potential modifier genes, one may consider that these phenotypes actually constitute a continuum. Nevertheless, different grades of severity can be attributed to the genetic heterogeneity of the disease, in particular when considering the respective roles of HC proteins in iron homeostasis. These grades range from the HJV and HAMP-related juvenile conditions to the typical adult-onset HFE-related condition, which is usually diagnosed in the fourth decade for men and the fifth for women. The iron overload course in TFR2-related patients is distinct from that of the juvenile conditions, but it can be more rapid and impressive than usually observed in HFE-related patients. Indeed, based on the review of the 23 TFR2-related cases reported in the literature until 2004 and with considering a cohort of 421 HFE p.C282Y/p.C282Y homozygous patients, we previously showed that age at diagnosis in TFR2-related patients was significantly lower than in HFE-related patients. We also highlighted the descriptions of clinical features (including arthralgy, hypogonadism, skin pigmentation and cirrhosis) in TFR2-related patients before the age of 30 years.4 Our observations have been reinforced by some recent reports, in which the identification of TFR2 mutations was correlated with the description of either high serum iron parameters in young patients or severe clinical pictures in older patients In a retrospective analysis of 33 cautiously selected Italian cases, Biasiotto and co-workers have more especially identified two children with new TFR2 causative mutations.9 The authors argued for a TFR2 genotyping in patients with early-onset of iron overload, and they further stressed that the TFR2-related HC is characterized by high transferrin saturation levels. Furthermore, mice homozygous for the p.Y245X TFR2 mutation (the murine ortholog of the human p.Y250X mutation 5 identified in the first TFR2-related pedigrees of HC) have proved to develop a more rapid hepatic iron overload than HFE knock-out mice of the same genetic background.10

In conclusion, it becomes more and more evident that TFR2-related HC is an intermediate syndrome between the typical adult-onset HFE-related HC and the two juvenile conditions. Thus, the search for TFR2 mutations should be particularly considered in young patients with haemochromatosis which do not exhibit a juvenile condition.