Hemochromatosis Gene Mutation and Erythrocytosis
In a patient group presenting with either increased Hgb level or high RBC count HFE gene mutations occured in two-third of the patients. These patients had normal erythropoietin levels and had no JAK2 mutation. Out of the 22 patients, 12 had the H63D mutation in heterozygous / homozygous or compound heterozygous forms, 2 had C282Y mutation and 7 had wild type HFE gene. In contrast to the classical form of type I Hemochromatosis with C282Y homozygous mutation, characterised by high serum iron, transferrin saturation and ferritin levels, patients with the H63D mutation had normal iron content. In the second part of the study authors analysed iron and red cell parameters of patients available from their Hemochromatosis registry. The results were similar: those who had the H63D mutation, had more red cells and less iron content. It seems like in those having this mutation more iron is consumed for the red blood cell production, but the explanation for this phenomenon is still missing. The role of H63D mutation in this process should still be clarified.
Introduction
Patients with high red blood cell count (RBC) and high hemoglobin concentration with janus kinase 2 (JAK2) mutation and low erythropoietin (EPO) level- represent the group of true polycythaemia (PV) and those with wt JAK2 and high EPO are the group of the so-called secondary polycythaemia forms [1]. Those significant number of patients, however who presenting either high haemoglobin or RBC levels and has no JAK2 mutation, and has normal EPO levels still remain a diagnostic challenge.
It has been noticed, that regular therapeutic phlebotomy –as the main treatment option for Hereditary Hemochromatosis (HH) patients- is well tolerated by them, meaning that anemia does not develop even in the first year of diagnosis, when 400 ml blood / occasion is drained many times until the goal ferritin level achieved and maintained thereafter. In contrast to this, for a voluntary blood donor five blood donations are permitted in a year to avoid the development of anemia. In a survey of voluntary blood donors, the ratio of H63D heterozygotes was 31% in the group of the so-called super donors who donate 30-107 occasions [2]. Whether the high iron availability of HH patients is enough to explain this extremely good compensation of blood loss? In this study authors discuss this problem based on literature data and their recent findings.
Materials and Methods
The first study population consisted of 22 (16 M / 6 F, mean age: 61,6 yrs) non selected, consecutive and naive subjects who had not phlebotomy or other therapy before, were not blood donors and the reason of enrollment was their high Hgb levels or RBC counts found on rutin laboratory analysis. They were JAK2 wt (confirmed also by gene sequence analysis) and had normal EPO levels.
The second study population consisted of 35 hemochromatosis patients (16 with homozygous C282Y ,- 11 with compound heterozygous,- and 8 with homozygous H63D mutation) from the Hemochromatosis patients registry of our institute. All were males. Female patient were excluded from this study because of their regular menstrual blood loss and also those males, who had known co-morbidities causing bleeding or bone marrow disorders with impaired red cell production, like myelodysplastic syndromes.
The participants signed informed consents, and the study was approved by the Institutional Ethics Committee. Whole genomic DNA was isolated from anticoagulated peripheral blood with Puregene Gentra DNA Isolation kit. HFE gene sequence variant C282Y (exon 4, c.1066G>A, substitution of Cys282 to Tyr, rs1800562); and H63D (exon 2, c.408C>G, substitution of His63 to Asp, rs1799945) were investigated by Light Cycler technology (Roche Diagnostics) applying melting curve analysis with the hybridization probe detection format [3]. The JAK2 gene V617F mutation (exon 12, c.1849G>T, substitution of Val617 to Phe) was detected by allele specific multiplex PCR [4].
Results
In the first study population serum iron, transferrin saturation and ferritin concentration did not differ from normal values, but red blood cell count / or hemoglobin was higher in those, who had in any type the HFE gene mutation Table 1.
| EPO | RBC | Hgb | Se Fe | Tf sat | Ferritin | |
|---|---|---|---|---|---|---|
| mU/ml | Tera/l | g/l | umol/l | (%) | ug/l | |
| HFE wt n=7 | 5,6 | 5,7 | 181 | 23,5 | 37 | 293 |
| HFE mut n=15 | 11,9 | 6,0 | 187 | 17,8 | 30 | 242 |
| Normal | 2,6-18,5 | F/M max | F/M max | F/M max | 16-45 | F/M max |
| 5,1/5,9 | 153/175 | 28/32,2 | 250/300 |
Table 1: Iron and red cell parameters of JAK2 wt patients with erythrocytosis having or having not HFE gene mutation.
Mean values are shown. The upper normal values are given for Females and Males. Table 1: Iron and red cell parameters of JAK2 wt patients with erythrocytosis having or having not HFE gene mutation.
| C282Y 1/0 | H63D 0/1 | H63D 0/2 | C282Y /H63D 01/01 | |
|---|---|---|---|---|
| HFE mutant (n:15) | 2 | 8 | 1 | 4 |
Table 2: HFE gene mutation results of the 15 patients with JAK2wt erythrocytosis.
Abbreviations: 1/0 or 0/1 are heterozygous, 0/2 are homozygous, 01/01: compound heterozygous. Table 2: HFE gene mutation results of the 15 patients with JAK2wt erythrocytosis.
contain the H63D mutation and the highest iron values in those, who had the C282Y in homozygous form Table 3.
RBC Terra/l Hgb g/l SeFe umol/l tfsat (%) ferritin ug/l compound
418 (31-1678) H63D
336 (114-547) C282Y
n= 16
- Mean values are shown. The upper normal values are shown in Table 1.
Table 3: Iron and red cell parameters of a Hemochromatosis patient population.
Discussion
In this preliminary study HFE gene mutation occurred in two-third of patients presenting either high Hgb or RBC count, but did not fulfil the criteria of PV. There was a male preponderance. Literature data confirm that HH genetic polymorphism is associated with more iron availability / and more iron absorption especially in a compound heterozygous form [5, 6]. Would this be enough to explain erythrocytosis? More iron mean more red cell or Hgb?. It has been shown that both types of TfRs bind to HFE proteins and have role in iron sensing [7]. TfR2 is recently found to be an EPO partner receptor [8]. Based on these findings we might explain erythrocytosis seen in Tfr2 knockout mice and in Type 3 hemochromatosis patients who tolerate well repeated phlebotomies. Authors of the present study therefore assumes that co- existent TfR2 gene mutation might hide in the background in JAK2wt/norm EPO / HFE – related or unrelated erythrocytosis, that is to be further analysed. Multiple pathways might exist influencing erythropoiesis other than EPO signaling mechanism. Among these could be- not surprisingly- genes participating in iron metabolism regulation that still should be explored.
Conclusion
The significant ratio of HFE gene mutation in the group of patients presenting with high red blood cell count and hemoglobin level outlines the importance to perform the test in all cases of unexplained (JAK 2 wt, normal level EPO) erythrocytosis.
Acknowledgements: Author is thankful to Ambrus Gángó, Richárd Kiss, Csaba Bödör, Dorottya Csuka and Márta Kókai for the genetic analysis.
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