|Year : 2013 | Volume
| Issue : 1 | Page : 36-40
Frequency of human hemochromatosis (HFE) gene mutations in Egyptians with β-thalassemia
Doha A Mokhtar1, Mona M Hamdy2, Ahmed M Badr2
1 Department of Clinical Pathology, Faculty of Medicine, Cairo University, Cairo, Egypt
2 Department of Pediatrics, Faculty of Medicine, Cairo University, Cairo, Egypt
|Date of Submission||20-Jul-2012|
|Date of Acceptance||13-Sep-2012|
|Date of Web Publication||20-Jun-2014|
Doha A Mokhtar
Department of Clinical Pathology, Faculty of Medicine, Cairo University, Cairo
Source of Support: None, Conflict of Interest: None
Hereditary hemochromatosis is a disorder of iron metabolism characterized by increased iron intake and progressive storage and is related to mutations in the HFE gene. Two point mutations have been described and are referred to as H63D and C282Y. On the other hand, iron overload is a well-documented complication in thalassemia syndromes. Interactions between thalassemia and hemochromatosis may further increase iron overload. This work aimed at studying the frequency of the H63D and C282Y mutations of the HFE gene in an Egyptian population with β-thalassemia (thalassemia major, intermedia, and minor) by comparing it with normal individuals without hemoglobinopathies.
Participants and methods
This study included 86 patients with β-thalassemia; 40 of these patients had β-thalassemia major and intermedia and the other 46 patients had β-thalassemia minor (carriers). In addition, 70 individuals were included in the study and served as controls. All the populations studied were screened for H63D and C282Y mutations of the HFE gene using the PCR-restriction fragment length polymorphism (PCR-RFLP) method.
The allelic frequencies found for H63D and C282Y mutations in this study were 18.6 and 0%, respectively, among the total alleles of individuals with β-thalassemia and 12.8 and 1.4% among controls without hemoglobinopathies. On comparing thalassemia cases, thalassemia carriers, and the control group, the allele frequency of the H63D mutation was significantly higher among β-thalassemia carriers (24%) compared with patients with β-thalassemia (12.5%) and controls (12.8%) (P=0.049). Our study also found an almost significantly higher frequency of the heterozygote genotype for the H63D mutation in patients with β-thalassemia (30.2%) than the controls (17.1%) (Fisher’s test, P=0.06). Compound heterozygosis for H63D and C282Y was found in one of the individuals in our control group.
The frequent occurrence of β-thalassemia disease with the H63D gene mutation raises the possibility of genetic interactions and emphasizes the value of screening for HFE mutations in thalassemias to detect early cases of iron overload and to modify its treatment modalities.
Keywords: hemochromatosis, HFE gene, β-thalassemia
|How to cite this article:|
Mokhtar DA, Hamdy MM, Badr AM. Frequency of human hemochromatosis (HFE) gene mutations in Egyptians with β-thalassemia. Egypt J Haematol 2013;38:36-40
|How to cite this URL:|
Mokhtar DA, Hamdy MM, Badr AM. Frequency of human hemochromatosis (HFE) gene mutations in Egyptians with β-thalassemia. Egypt J Haematol [serial online] 2013 [cited 2020 Apr 10];38:36-40. Available from: http://www.ehj.eg.net/text.asp?2013/38/1/36/134801
| Introduction|| |
Hereditary hemochromatosis (HH) is an iron metabolism disorder characterized by increased iron absorption and storage, resulting in progressive and multisystemic oxidative organ damage 1,2. In 1996, the human hemochromatosis (HFE) gene was identified on chromosome 6 and considered to be a candidate for the gene bearing the primary defect responsible for hemochromatosis 3. A missense mutation that changes the 282 amino acid from cysteine to tyrosine (C282Y) has been characterized by Feder et al. 3 as the main mutation responsible for hemochromatosis. The C282Y mutation disrupts a critical disulfide bond in the a3 domain of the protein, preventing the HFE molecule from interacting with β2-microglobulin 4,5. A second mutation has also been described in HH patients 3, changing the aspartic acid for histidine at position 63 of the protein (H63D). Although this mutation does not affect the protein cell surface expression, its ability to reduce the HFE–transferrin receptor interaction may explain its relation to iron overload 5.
However, the phenotypic expression of mutated alleles seems to be highly variable and is possibly related to other coinherited genetic modifiers, including genes related to hereditary anemia 6,7.
In Egypt, β-thalassemia is the most common genetically determined, chronic, hemolytic anemia, with an estimated carrier rate of 9–10.5% 8.
When anemia is accompanied by increased erythroid activity and/or ineffective erythropoiesis as in thalassemias 9, there is a concomitant increase in the absorption of iron from the diet because of higher iron needs for the synthesis of hemoglobin. These patients develop iron overload even without erythrocyte transfusions. If transfusions are required, they will add to the body iron excess. The interaction of the HH mutations with the thalassemias may have a synergistic effect, increasing the iron intake and storage 10,11.
Our aim of this work was to study the frequency of the H63D and C282Y mutations of the HFE gene in a group of Egyptian population with β-thalassemia (thalassemia major, intermedia, and minor), comparing it with normal individuals without hemoglobinopathies.
| Participants|| |
This study included 86 patients [33 (38.4%) males and 53 (61.6%) females] with β-thalassemia syndromes 40 of these patients had β-thalassemia major and intermedia who were regularly attending the Hematology Clinic of the New Children Hospital, Cairo University (their ages ranged from 0.5 to 16 years with a median age of 3 years), and the other 46 patients had β-thalassemia minor (β-thalassemia carriers) who were mostly selected from the parents of thalassemia patients not included in our study (their ages ranged from 23 to 39 years with a median age of 31 years). In addition, 70 individuals [33 (47.1%) males and 37 (52.9%) females] were included as a control group; their ages ranged from 2 to 35 years, with a median age of 8 years. The diagnosis of β-thalassemia was made on the basis of clinical presentation, hematological indices, and hemoglobin electrophoresis or HPLC. For β-thalassemia major, all patients were on regular blood transfusion regimens, whereas for β-thalassemia intermedia, patients received no or sporadic blood transfusions throughout their lives. The diagnosis of β-thalassemia minor was defined on the basis of red cell microcytosis [mean corpuscular volume (MCV) <80 fl] and hypochromia [mean corpuscular hemoglobin (MCH) <27 pg], and confirmed by elevated hemoglobin A2 (>3.5%). There were three patients with sickle/thalassemia in our studied populations.
Hematological indices and hemoglobin characterization for different thalassemia groups are presented in [Table 1]
|Table 1: Hematological characterization of different thalassemia groups included in our study|
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An informed consent approved by the Institutional Ethics Committee was obtained from all participants or their parents.
| Methodology|| |
Blood samples were collected by sterile venipunctures on EDTA vacutainer tubes that were stored at −20°C for subsequent DNA extraction and further genotyping.
HFE gene mutation testing
Genomic DNA was extracted from whole blood of all patients and controls using the QIAamp DNA Kit (catalogue number 51104; Qiagen, Hilden Germany). Detection of the mutations H63D and C282Y in the HFE gene was carried out by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis according to Merryweather-Clarke et al. 12. The two regions of the gene containing the proposed mutations were amplified by PCR before RFLP analysis. For both PCR products, primers were designed to include an internal restriction enzyme site in the product to act as a control for complete digestion. Both PCR reactions were performed under the following thermocycling conditions: an initial denaturation (94°C, 5 min), followed by 35 cycles of denaturation (94°C, 30 s), annealing (58°C, 30 s) and extension (72°C, 30 s), and then a final extension (72°C, 5 min). For the H63D mutation, the following primers yielded a product of 294 bp: H63DF: 5′ ACA TGG YEA AGG CCT GTT GC; H63DR: 5′ CGT GCT GTG GTT GTG ATT TYC C. Following digestion with MboI (FastDigest MboI; Fermentas, Germany), those products carrying the mutation yielded restriction fragments of 237 and 57 bp, whereas fragments lacking the mutation contained an extra MboI site and yielded restriction fragments of 138, 99, and 57 bp [Figure 1]. For the C282Y mutation, the following primers yielded a product of 343 bp: C282YF: 5′ CAA GTG CCT CCT TTG GTG AAG GTG ACA CAT; C282YR: 5′ CTC AGG CAC TCC TCT CAA CC. Following digestion with RsaI (FastDigest RsaI; Fermentas, Germany), those fragments containing the mutation carried an additional RsaI site, resulting in products of 203, 111, and 29 bp, whereas those lacking the mutation yielded products of 203 and 140 bp.
| Results|| |
The genotype frequencies of H63D and C282Y mutations are presented in [Table 2]
|Table 2: Genotype frequency of both H63D and C282Y gene mutations among thalassemia patients and controls|
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Among the 86 β-thalassemia patients studied, 57 (66.3%) and 86 (100%) carried the wild-type profile, 26 (30.2%) and 0 (0%) carried the heterozygous genotype, and 3 (3.5%) and 0 (0%) carried the homozygous mutant genotype for H63D and C282Y mutations, respectively.
Of the 70 healthy controls studied, 55 (78.6%) and 68 (97.1%) carried the wild-type profile, 12 (17.1%) and 2 (2.9%) carried the heterozygote genotype, and 3 (4.3%) and 0 (0%) carried the homozygous mutant genotype for H63D and C282Y mutations, respectively. One of the two C282Y heterozygotes also carried the heterozygous H63D genotype.
Our study showed an almost significantly higher frequency of the heterozygote genotype for the H63D mutation in β-thalassemia patients than the controls (Fisher’s test, P=0.06).
Comparison of three groups of β-thalassemia patients, β-thalassemia carriers, and controls without hemoglobinopathies indicated a significant difference between them in the H63D gene mutation; the wild genotype frequency was 32 (80%), 25 (54.3%), and 55 (78.6%), the heterozygote genotype frequency was 6 (15%), 20 (43.5%), and 12 (17.1%), and the homozygous mutant genotype frequency was 2 (5%), 1 (2.2%), and 3 (4.3%) for thalassemia patients, carriers, and controls, respectively (P=0.011) [Table 3].
|Table 3: Comparison between β-thalassemia patients, β-thalassemia carriers, and controls without hemoglobinopathies in the genotype frequency of the H63D gene mutation|
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No significant association was found between genotype frequency and sex distribution among cases and controls, which is shown in [Table 4].
|Table 4: Association between the genotype frequency of both H63D and C282Y gene mutations and sex distribution among cases and controls without hemoglobinopathies|
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The allelic frequencies obtained for the HFE gene mutations H63D and C282Y were (18.6 and 0%), respectively, among the total alleles of β-thalassemia patients and (12.8 and 1.4%) among controls without hemoglobinopathies. These data are shown in [Table 5].
|Table 5: Allele frequencies of H63D and C282Y mutations among total alleles of β-thalassemia individuals and controls without hemoglobinopathies|
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The allelic frequencies of the H63D gene mutation among the total alleles of β-thalassemia patients, β-thalassemia carriers, and controls without hemoglobinopathies are presented in [Table 6]. There was a significantly high allelic frequency of the H63D mutation among β-thalassemia carriers (24%) compared with cases (12.5%) and controls (12.8%) (P=0.049).
|Table 6: Allelic frequencies of the H63D gene mutation among total alleles of β-thalassemia patients, β-thalassemia carriers, and controls without hemoglobinopathies|
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Data obtained from the study were coded and entered using the software SPSS (statistical package for social science; SPSS Inc., Chicago, Illinois, USA) version 11. Parametric data were summarized using mean and SD, whereas nonparametric data were summarized as median and percentiles for quantitative variables. Frequency and percentages were used for qualitative variables. Comparison between groups was carried out using the χ2 and the Fisher’s exact test for qualitative variables; the t-test and a nonparametric Mann–Whitney test were used to compare two groups, whereas analysis of variance and a nonparametric test (Kruskal–Wallis test) were used to compare multiple groups. P value less than 0.05 was considered significant.
| Discussion|| |
The allelic frequencies found for H63D and C282Y mutations in this study were 18.6 and 0%, respectively, among the total alleles of β-thalassemia patients and 12.8 and 1.4% among controls without hemoglobinopathies. These results are consistent with the worldwide prevalence of the H63D mutation and the North European restriction of the C282Y mutation. Our results are almost the same as the values obtained by Panigrahi et al. 13 in 2006, who reported allelic frequencies of the H63D mutation of 22.5% among Asian Indian patients and 8.1% among controls. The frequency of the C282Y mutation was 0% among patients and controls. Our results are also in agreement with a screening study for hemochromatosis mutations in Iranian β-thalassemia minor patients, where the H63D and C282Y allele frequencies were 12.9 and 1.6% in patients compared with 8.7 and 0% in controls, respectively 11. Martins et al. 14 also studied the allelic frequencies of the above two gene mutations among β-thalassemia carriers in Portugal compared with a control group, and their frequencies were 15.3 versus 18.3% for the H63D mutation and 1.5 versus 3.5% for the C282Y mutation. These results are also in agreement with those of Oliveira et al. 15, who reported allelic frequencies of the H63D and C282Y mutations among Brazilian β-thalassemia carriers of 13.7 and 2.4% compared with 9.5 and 0.3%, respectively, among controls. In agreement with our results, earlier Egyptian studies have reported that the allele frequency of the H63D mutation ranged from 13 to 30% in thalassemic patients and between 10 and 11% in controls 16.
The difference between H63D and C282Y allele frequencies in all of our study population (both cases and controls) was in agreement with that reported previously by Merryweather-Clarke et al. 12, who found worldwide allele frequencies of 1.9% for C282Y and 8.1% for H63D, with the highest frequency of C282Y in northern European populations, and its absence from African, Asian, and Australasian chromosomes included in their study. However, the H63D is known to have a wider distribution, with a higher allele frequency being observed in countries around the Mediterranean, decreasing in populations with distance from the Mediterranean region 17. Our results in this issue are almost in agreement with those of other studies in Africa showing an absence of the C282Y mutation in Algeria, Ethiopia, and Senegal. The H63D mutation, although absent in Senegalese, was found in about 9% of the chromosomes genotyped among the central Ethiopians and Algerians 18. The prevalence of these two mutations was also detected among Tunisian populations and their allelic frequencies were 15.17 and 0.09% for H63D and C282Y mutations, respectively 19. Also, our genotype frequencies were in agreement with the results of a study carried out in Egypt by Settin et al. 20, who reported H63D and C282Y genotype frequencies to be 21.2 and 0.0%, respectively, in their controls.
On comparing thalassemia patients, thalassemia carriers, and the control group, the allele frequency of the H63D mutation was significantly higher among β-thalassemia carriers (24%) compared with β-thalassemia patients (12.5%) and controls (12.8%) (P=0.049).
This result is in agreement with that of Jazayeri et al. 11, who found significant differences in the frequencies of H63D and C282Y mutants among β-thalassemia carriers in relation to control individuals; however, Martins et al. 14 found no significant differences between β-thalassemia carriers and controls in the allelic frequencies of the above gene mutations. In contrast, Oliveria et al. 15 found a significant difference among β-thalassemia minor and control groups in the C282Y mutation, but not the H63D mutation.
Our study also found an almost significantly higher frequency of the heterozygote genotype in the H63D mutation in β-thalassemia patients (30.2%) than the controls (17.1%) (P=0.06). In another study carried out by Sharma and colleagues, a frequency of 25.8% heterozygous for H63D was found among patients of thalassemia intermedia included in their study, whereas the prevalence of H63D heterozygosity was 7.5% among healthy controls. They observed that all patients had serum ferritin greater than 500 ng/dl and concluded that thalassemia intermedia patients with a coexistent HFE mutation have a higher likelihood of developing iron overload and may require early iron chelation 21. However, Oliveria et al. 15 found no significant difference in the heterozygote genotype frequencies among their patients and controls.
Compound heterozygosis for H63D and C282Y was found in one of our control group populations who carried both mutations. This compound heterozygosity seems to predispose to disease expression 7.
Although the β-thalassemia trait is characterized by mild, ineffective erythropoiesis that can induce excess iron absorption, only a few patients develop iron overload; the hypothesis that genetic modifiers may be related to this phenotypic variability has been put forward. Among these, HFE gene mutations seem to be the most probable cause 22.
Two independent pathways have been proposed for iron metabolism: the erythroid regulator, which modulates intestinal iron absorption in response to the needs of the erythron, and the storage regulator, which controls iron accumulation 23,24. There are suggestions that the erythroid regulator (β-thalassemia) seems to be more pronounced than the storage regulator (the mutated HFE gene) in determining the degree of iron absorption 23,25. This hypothesis indicates that β-thalassemia carriers might exhibit an advantage in balancing iron storage in their organisms. Nevertheless, published data indicate more severe symptoms of hemochromatosis when heterozygosis for the C282Y mutation is associated with β-thalassemia 10, and when higher serum iron levels in β-thalassemia coinheritance are associated with heterozygosis and homozygosis for H63D 6,26.
In our Egyptian population, iron deficiency was very common because of deficient intake. This concomitant iron deficiency or low iron stores would protect against iron overload in patients with the β-thalassemia trait with a mutation in the HFE gene as previously described by Garawel et al., in 2005 27. This may not be true in populations with a dietary intake of sufficient or high iron content. Therefore, iron overload in patients with the β-thalassemia trait may be population specific and the causative mutations or polymorphism may vary between different populations 27.
| Conclusion|| |
Our study has shown that, of the two common mutations, H63D and C282Y in the HFE gene, only H63D was found to be more frequent in β-thalassemia carriers than controls and the frequency of the heterozygous H63D genotype is higher in β-thalassemia individuals than controls. The frequent occurrence of β-thalassemia disease and the H63D gene mutation raises the possibility of genetic interactions and emphasizes the value of screening for HFE mutations in thalassemia to detect early cases of iron overload and to modify its treatment modalities.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]