Biallelic Mutations in Hamp in Type 2b Hemochromatosis

Introduction

Hereditary hemochromatosis (HH) is a group of genetic iron overload disorders caused by mutations in genes encoding HFE (HFE1), transferrin receptor 2 (TFR2), hepcidin (HAMP), hemojuvelin (HFE2) or ferroportin (SLC40A1), respectively. HH is classified into 4 different categories according to the gene causing the disease. Type1 is the most common form arising from homozygosity for HFE C282Y mutation at the homozygous state. Type2 HH includes type 2A caused by mutations in HJV and type 2B , caused by mutations in HAMP. Hepcidin is synthesized as a pro-peptide of 84amino acids, which is cleaved into peptides of 20, 22, and 25 amino acids, the last being the active form of hepcidin. It is established that hepcidin negatively regulates iron export by direct interaction with ferroportin. Mutations or deficiency in Hepcidin result in excessive iron absorption that can lead to massive iron overload. We report a patient with a severe iron overload related to an unusual genotype of two previously unreported HAMP variants in combination with the H63D HFE mutation.

Case report:

A 69 year old male patient was diagnosed to have a severe iron overload with serum ferritin (SF) 5076 µg/l , transferrin saturation (TS) 48% and liver iron concentration (LIC) 290 µmolFe/g. Factors contributing to the hyperferritinemia included a chronic daily consumption of alcohol (100 g /days with gGT : 150 UI/l). Genetic analysis revealed heterozygosity for the common HFE1 H63D mutation and two new mutations in HAMP: one located in the promoter ( -274 G>C ) and the other in the stop codon (c. 255 G>C , p. Ter85TyrextTer21) . After one year of phlebotomy therapy, 5.5 g iron was removed and serum ferritin dropped to 1700 µg/L. Phlebotomy therapy was interrupted when patient was lost to follow-up and one year later SF was 2730 µg/L. Hepcidin was measured using mass spectrophotometry before resuming the phlebotomy (27.6 ng/ml, nle : 1 to 20 ng/ml).

Methods :

In order to analyze the involvement of the two HAMP mutations in the iron overload, further DNA studies were performed including MLPA (MRC Holland , P347 Hemochromatosis probemix , version A2) and Next generation sequencing (NGS, Miseq^TM, Illumina ). The iron regulatory gene panel included the following gene sets : HFE-1; HFE-2 ; TFR2; HAMP; SLC40A1; FTH1; TF; FTL; SLC11A2; TMPRSS6; HEPH; CP; ALAS2; BMP6;BMP4; SMAD4. The Hepcidin functional tests were performed by using HEK293T cells expressing FPN1. Subsequently, cells were grown with preconditioned supernatants obtained from cells transfected with hepcidin^wt or hepcidin^255C. Promoter mutation was tested using HepG2 cells lines. Linkage between the two mutations was tested by means of DDPCR (Bio-Rad, QX200™ Droplet Digital™). We used simultaneously two of hydrolysis probes with different labeling and results were analyzed by a Poisson statistical comparison of double labeled drops count versus simple labeled drop.

Results of these studies showed that :

1- No other significant mutation could be retrieved with the NGS analysis and MLPA analysis of HFE, HFE2, HAMP, TFR2 and SLC40A1 did not show any pathogenic copy number variant.

2- The elongated hepcidin was unable to induce internalization of ferroportin.

3- The elongated hepcidin did not display any dominant effect (ie: unability to suppress the effect of wt hepcidin)

4- The functional study of the -274 G>C promoter mutation fails to show a reproducible and significant decreased function compared to wt

5- The two mutations were located in trans

Discussion and conclusion.

The reason why in some HAMP-P individuals iron overload was comparable to that of wild-type patients could be due to the fact that they are heterozygous for the SNP and, therefore, show an incomplete penetrance or compensatory mechanisms. We report a patient in which the only iron metabolism related mutations are the Hamp:c.255 G>C (p. Ter85TyrextTer21 mutation) and an SNP in the promoter: Hamp:c.-274 G>C mutation. We show that the elongated hepcidin is a loss of function mutant. Unfortunately, we could not have access to other family members for the segregation study of the HAMP mutations. Serum Hepcidin level was determined when ferritin was 2730 µg/l and appears to be low according to iron parameters. Our observations suggest a hypothetical role of the HAMP-P -274 G>C SNP on promoter reactivity. From this set of data, we propose that the patient is affected by a new Hamp linked HH (HFE 2B) probably worsened by drinking habits.