The phenotypic diversity of beta (β)-thalassemia is associated with genetic modifiers and environmental factors. Primary genetic modifiers are the wide range of β-globin mutations principally affecting β-globin chain synthesis. Secondary modifiers also affect β-globin synthesis through variations in the alpha (α)-globin or gamma (γ)-globin synthesis. Tertiary modifiers are not associated with β-globin synthesis but alter the complications of the disease. Tertiary modifiers include distinct genetic polymorphisms co-selected with β-thalassemia, further adjusting the phenotype by modifying the complications. Some of these recognized complications are iron overload, hyperbilirubinemia, and osteoporosis (1–3).

In patients with transfusion-dependent thalassemia, iron overload following 2–3 years of initiation of blood transfusions is inevitable. Each 500 ml of packed red cell contains 250 mg of elemental iron, and repeated transfusions will saturate the available transferrin level in the circulation, favoring non-transferrin bound iron (NTBI), a toxic compound to be formed. This NTBI generates highly reactive hydroxyl radicals resulting in oxidative damage to various cellular components, such as lipids, proteins, and nucleic acids, causing tissue destruction (4–7). Hepcidin, a small peptide hormone synthesized by the liver, is the primary regulator of iron movement into plasma. When the hepcidin level is very low or absent in the plasma, as in iron deficiency anemia, iron is diverted to plasma from enterocytes and macrophages through the ferroportin transport mechanism (4, 8). Since β-thalassemia major patients have low hepcidin levels in their circulation, it results in an increased level of plasma free iron; iron overload, triggering tissue damage (4).

The coexistence of HFE gene-associated hereditary hemochromatosis and β-thalassemia can exacerbate iron overload and iron-related complications in patients with β-thalassemia. It is observed that hemochromatosis is frequently associated with β-thalassemia. Several studies have revealed that the interaction of hereditary hemochromatosis with β-thalassemia can have an exaggerated response in iron absorption and storage in these patients (9–16). In patients with HFE-associated hereditary hemochromatosis, two common missense mutations; c.845G>A (p.C282Y; rs1800562) and c.187C>G (p.H63D; rs1799945), have been described. There is a significant ethnic variation observed in the distribution of these variants; the p.C282Y mutation is mainly limited to the North European region. The prevalence of this variant is considered very low in Australian, African, and Asian populations. The p.H63D mutation is shown to have a cosmopolitan distribution across the world with a frequency of 3.3%−15.2% (17–19).

The degree of iron overload can be evaluated either by assessing serum ferritin levels or liver iron concentration (LIC). Assessing LIC using liver biopsy is the gold standard method and the most reliable body iron assessment indicator. Yet its invasive nature with potential morbidity and mortality (<1 in 10,000 cases), poor patient compliance, and sampling error have hindered its routine clinical use (14, 20–23). Serum ferritin levels generally represent body iron stores and have shown to be a convenient and reliable method to assess body iron stores. Furthermore, serial measurements help determine trends of the iron overload (24). However, serum ferritin is an acute-phase reactant and is elevated non-specifically in acute or chronic inflammatory states (25) and, therefore, is an unreliable predictor of body iron stores in the presence of inflammation. Other markers of inflammation, such as C-reactive protein (CRP), would help to eliminate this confounding factor when assessing body iron stores (26, 27). Present-day magnetic resonance imaging (MRI)-based techniques are the most extensively used techniques for LIC estimation. Universal unavailability and economic constraints have resulted in the underutilization of MRI-based assessment of body iron stores in routine clinical practice (28, 29). Serum glutamine aspartate transaminase (SGPT) is a hepatocyte-specific enzyme. It is released into the bloodstream following hepatocyte injury and, therefore, is routinely used as a marker of liver disease (30).

The objective of this study was to genotype p.C282Y and p.H63D variants of the HFE gene in transfusion-dependent β-thalassemia and HbE/β-thalassemia patients and correlates the mutation status with their serum ferritin levels.

Of the total study population of 125, 60 (48%) patients were male; the male to female ratio was 60:65. The study cohort consisted of transfusion-dependent 123 β-thalassemia patients and 2 HbE/β-thalassemia patients. The mean age of the study cohort was 8.86 years (SD ± 4.7), and the age range varied between 9 months to 23.5 years. Thirty-four (27%) patients in the study cohort were over 18 years.

Analysis of a three-generation pedigree revealed that 33 (26.4%) patients had third-degree parental consanguinity. However, the majority (67.2) did not have a history of consanguinity. The family history of consanguinity was not recorded in eight (6.4%) patients. The molecular diagnosis was available in 94 (75%) patients. HBB genotype distribution and their frequencies are shown in Table 1. The molecular diagnosis was not available in 31 (25%) patients (Table 1).

In most patients (58%), blood transfusion was initiated at 6 months. The range of time taken to initiation of the transfusion regimen was between 2 months and 2 years. The mean pre-transfusion hemoglobin level of this study cohort was 8.82 g/dl (SD ± 0.85), and a majority received their monthly blood transfusion when the pre-hemoglobin level was 8.7 g/dl. Of the 125 patients, 17 (13.6%) had undergone splenectomy at their early ages, and cholecystectomy had been performed in one (0.8%) patient.

The mean serum ferritin level was 4,628.5 ng/ml (SD ± 2,614), and the ferritin level ranged from 157 to 12,470 ng/ml. Serum ferritin reports of four patients were not available for analysis. Complications related to iron overload were common in the study group; seven (5.6%) patients had diabetes mellitus at recruitment. Hypothyroidism was reported in five (4%) patients, and one (0.8%) patient had hypoparathyroidism. Two (1.6%) patients in the study cohort had both diabetes mellitus and hypothyroidism. The CRP level was high in nine (7.2%) participants; the mean CRP was 2.2 mg/L. The ESR was elevated in 65 (52%) patients; the mean ESR was 21.7 mm/1st h. The SGPT was elevated in 82 (65.6%), and the mean SGPT was 91 U/L.

None of the study participants carried the p.C282Y variant allele. All study participants (100%) were the wild-type variant (c.845GG). The p.H63D variant allele was detected in the heterozygous state [c. 187CG] in 23 (18.4%) patients and allele frequencies were c.187CC – 91.8%, c.187CG – 9.2%, and c.187GG – 0%. The genotype distribution was in accordance with Hardy–Weinberg equilibrium (32). The agarose gel image results are shown in Figures 1, 2.

The mean serum ferritin levels were compared between the patients with the wild-type allele for p.H63D [c. 187CC] and the variant allele for p.H63D [c. 187CG]. Although the mean serum ferritin levels were high in both groups, the difference was not statistically significant (p-value = 0.865; Figure 3).

The comparison of the mean serum ferritin levels with the genotyping data is depicted in Table 2.

Of the nine (7.2%) patients with elevated CRP, four (3.2%) patients carried the wild-type variant (c.187CC) of the p.H63D mutation, while five (5%) participants carried the mutant variant (c.187CG). Of the 65 (52%) patients with high ESR, 50 (40%) participants carried the wild-type variant of the p.H63D mutation, and 15 (12%) had the mutant allele. Eighty-two (65.6%) patients who had high SGPT, 64 (51.2%) had the wild-type variant, and 18 (14.4%) had the mutant variant of the p.H63D mutation.

This study aimed to determine the allele frequency of the common variants p.C282Y and p.H63D in the HFE gene in a cohort of transfusion-dependent thalassemia patients and to determine the genotype–phenotype correlation between the variant status and body iron stores.

None of the patients in our study cohort harbored the p.C282Y (c.845G>A) variant; hence the allele frequency was 0%. This finding was similar to other populations in Asia (17, 33). Regarding the p.H63D (c.187C>G) variant, of the total study cohort, 23 patients were heterozygous for the variant allele (CG); hence the allele frequency was 9.2%. The prevalence of the c.187C>G variant had previously been analyzed in Sri Lankan cohorts. Following analysis of 109 chromosomes (218 alleles), Rochette et al. (33) reported the allele frequencies of p.C282Y and p.H63D variants as 0.8 and 10.8%, respectively. Rochette and his colleagues further reported a single case, a compound heterozygote for both mutations. After evaluating 130 referrals sent from Sri Lanka for hemoglobinopathy diagnosis, Merryweather-Clarke concluded that the p.C282Y mutation was absent on the island, and the frequency of the p.H63D mutation was 9.2% (34) (Table 3).

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.

The studies involving human participants were reviewed and approved by Ethics Review Committee (ERC) of the Faculty of Medicine, University of Colombo, Sri Lanka. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.

All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.

This study was supported by the NOMA scholarship to follow the MSc in Clinical Genetics funded by the Norad and managed by the Centre for International co-operation in Higher Education, Oslo, Norway.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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