Iron overload remains to be a major complication among the frequently transfused β-TM patients, especially if patients are not compliant to the iron chelation therapy. The demographic data of the present study illustrates a high burden of Iron overload in Egyptian β-TM patients as the mean serum ferritin was 4566.6 ± 3287.3ng/ml, this high value was a.

The local Iron tissue accumulation assessed by MRI of the liver and heart showed that the mean LIC of the patient’s group was 16.4 ± 8.40 mg/g, while the mean patient’s cardiac T2* was 29.69 ± 14.66 ms. These findings highlight the need to improve the implementation of patients follow up and education programs among the Egyptian β-TM to fulfill adequate patient compliance to iron chelation therapy, as unfortunately 72 patients (74.2%) of our β-TM patients were non-compliant to the iron chelation therapy.

Another challenging consequence of the recurrent blood transfusion that faces the health care system is the prevalence of hepatitis C viral (HCV) infection. In our study, 28 patients (29%) were positive for HCV infection; this is lower than the incidence reported among Egyptian β-TM in 2016 (37.11%) 10 which in turn is less than the incidence reported in 2012(69%) 11.

In 2008, Egypt had the highest burden of HCV infection worldwide. In 2015, the seroprevalence of HCV infection in Egypt has declined to 6.3% 12 we believe that this would be reflected as a lower HCV incidence among β thalassemia patients in future studies.

To the best of our knowledge, the prevalence of feroportin-1(-8CG), TMPRSS6 (rs855791) and HJV (I222N & G320V) gene polymorphisms in β-TM patients and the study of their possible relation to the development of iron overload was not previously studied but the effect of those genotypic variations was investigated in other conditions of altered iron metabolism.

This study revealed that heterozygous FPN1 C/G genotype was statistically significantly higher in patients (34%) when compared to the controls (16%), the wild C/C genotype was detected in 80% of the control subject and 62.9% of the cases, while 2 control subjects and 3 cases harbored the homozygous GG genotype with no statistical difference.

No significant relation could be detected between the FPN1 -8CG polymorphism and the level of serum ferritin denying the possibility of its contribution to increasing the soluble systemic iron stores however local liver iron reflected by the MRI LIC was significantly higher in patients harboring the (GG) FPN1 genotype when compared to those with the wild type (CC) genotype (p value = 0.03) suggesting the possible role of the FPN1 -8CG polyomorphism in enhancing the local iron overload within the liver. After statistical adjustment of other variables, we found that FPN1gene mutation acts as independent predictor of MRI LIC (p = 0.011).

Also, PAP was significantly higher in patients harboring the (GG + GC) FPN1 genotype in comparison to those with the wild (CC) genotype (p value = 0.048) suggesting the role of this mutation in producing local effects on the cardio vascular functions.

Ferroportin is the only known cellular iron exporter. It is regulated at a post-translational level by the main iron regulatory peptide; hepcidin. Decreased ferroportin expression would reduce external iron export and maintain intracellular iron 13. The FPN1 -8CG position is extremely close to the iron responsive element (IRE) of the gene, thus potentially interfering with FPN1 expression 14.

Despite being not previously studied among β-TM patients, FPN1 gene mutations have been investigated in other conditions of tissue iron overload. Castiglione et al., in 2015 studied the possible role of FPN1 − 8CG Polymorphism in modifying the iron homeostasis at the local level where they found that the homozygous genotype FPN1 − 8GG was significantly associated with increased risk of developing sudden sensory-neural hearing loss. It was assumed that increased levels of Fe2 + inhibit the intracellular production of nitrous oxide, a fundamental element in the regulation of local microcirculation 15.

Gemmati et al., 2009 studied the FPN − 8CG SNP and reported that patients harboring the (GG) genotype had a significant 5 folds increase in risk of acquiring chronic venous ulcer, this could be attributed to alteration of the macrophage membrane interfering with the efflux of iron from inside the cell with consecutive increase in oxidative stress 14.

Another study for the same SNP was conducted in Multiple sclerosis (MS) patients and concluded that there was a 4 folds increased risk of MS associated with the FPN1 -8GG homozygous genotype. In addition, disease progression and severity gradually increased as the number of the G alleles increased 16.

This however doesn’t agree with the results obtained by Tisato et al., were it was found that FPN1 -8G allele was under represented in vascular dementia patients although iron-driven oxidative stress is a key recognized physiopathological contributor for the disease, yielding a significant 1.5 fold risk reduction 17.

Regarding the TMPRSS6 genotypes, in this study, the homozygous TMPRSS6 (TT) genotype was statistically significantly lower in patients (41.2%) when compared to the controls (50%). The iron lowering allele, T allele, in homozygous state (TT) or heterozygous state (TC) was significantly lower in β-TM patients when compared to the control group (p = 0.042 and 0.049 respectively). This could point to a role of this genotype in iron overload pathogenesis and expression.

A study for the effect of the TMPRSS6 rs855791 polymorphism and consequent increase in hepcidin release concluded that TMPRSS6 polymorphism is likely a modifier of hereditary chromatosis (HH) expression 18. Also, a systematic review performed by Gichohi-Wainaina et al. in 2015 with meta-analysis done on TMPRSS6 loci identified in cohorts of differing ethnicity, they concluded that the T allele of rs855791 was associated with lower Hb and ferritin concentrations in all populations. This allele was also associated with increased serum transferrin receptor and transferrin concentrations 19.

A study carried out on the TMPRSS6 rs855791 polymorphism in the Egyptian population in 2018 showed a significant increase in frequency of mutations (TT and TC) in the counterpart disease of iron deficiency anemia patients compared to the normal group 20.

Hemojuvelin (HJV) is another protein that plays a crucial role in the regulation of hepcidin 21.

In the current study, no significant difference was found between the patients and control groups regarding the HJV 1222 N and HJV G320V genotypes frequencies (p = 0.831 and 0.774 respectively).

The present work showed significantly lower T2 among β-TM patients harboring the HJV 1222 N mutant variants reflecting higher cardiac iron concentration (p = 0.026). However LIC did not differ significantly among β-TM patients having various HJV 1222 N SNP genotypes.

Moreover, LVPWT was significantly higher in patients harboring the variant HJV1222N genotypes compared to those with the wild type (p value = 0.030) suggesting a higher degree of cardiac impairment in this group.

The current study revealed that 3 β-TM patients (3.1%) and 2 control subjects (4%) harbored the mutant allele for HJV G320V SNP.

Some studies have reported that the HJV gene mutations are uncommon.22, 23. The relative higher prevalence of those rare genotypes in the Egyptian population could be attributed to the higher incidence of consanguinity in marriage 24.

Previous studies have reported that HJV gene polymorphisms act as genetic modifiers in the hereditary hemochromatosis (HH) phenotype. A study in 2004 reported that association of heterozygous mutations in the HFE and HJV mutations could lead, at least in some cases, to an adult-onset form of primary iron overload 25 however this does not agree with the results obtained by a gene sequencing study conducted in 2010 26, also Alte`s et al. in 2009 found a minor impact of these mutations in the development of HFE-related hemochromatosis 27.

In conclusion this study identified that FPN1gene mutation could be considered as a reliable independent predictor of MRI LIC (p = 0.011), in addition the PAP was significantly higher in patients harboring the mutant allele of the same SNP.

Although this study results need to be confirmed by larger cohorts of patients with longer follow-up periods, our results suggest that established genetic risk factors in β-TM might cause different clinical phenotypes and thus make patients differently suited to iron over load management strategies.