The present study was set up to analyze the association of TMPRSS6 rs855791 polymorphism with iron deficiency among a Sri Lankan cohort of pregnant women. As of present, there were no studies conducted in Sri Lanka to identify the genetic predisposition of iron deficiency (ID) among any risk group, despite the observed high prevalence of ID within the population. In line with several previous research [20,21,22,23] conducted on populations in other parts of the world, our study indicated an increased risk of development of ID associated with the rs855791 polymorphism. In addition, the present study suggests for the first time, a possible mechanism through which the observed effect of iron regulation is being exerted by rs855791 polymorphism using an in silico protein binding model.
According to the results obtained, the T allele of rs855791 of TMPRSS6 gene predominates among the study cohort. This is comparable to what had been reported previously for other South Asian populations in which T allele predominates with a frequency around of 0.54 (ref: dbsnp https://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?do_not_redirect&rs=rs855791 and ). On the other hand, in African countries, the C allele predominates with a frequency ranging from 0.82 to 0.93 . This heterogeneity in allele frequency distribution observed for rs855791 TMPRSS6 gene among different ethnicities is suggestive of its significant functional role in different populations. For example, the high prevalence of the C allele in African countries in contrast to Asians and Europeans may be an adaptation to counteract severe famine. Given its higher potency to inhibit hepcidin transcription, the C allele might be advantageous in increasing iron absorption when dietary iron availability is severely limited .
The current study population represents a cross section of apparently healthy pregnant women who visited the antenatal clinics within Colombo municipal council area. Among them, a significant percentage (69.8%, 51/73) was iron deficient according to their corrected (for hs-CRP) serum ferritin levels as reported earlier . This highlights the need for immediate attention of the public health authorities in mitigating iron deficiency among pregnant women with a similar socioeconomic background. According to our study results, iron deficiency could have stemmed from two sources: a genetic propensity of the study population toward iron deficiency which is mediated via the rs855791 SNP and the observed low dietary iron intake.
Among our study population, iron deficient subjects showed a high propensity to carry T allele (61.8%) with more than twofold enhanced risk for iron deficiency. Having TT genotype increased the risk further up to sixfold. The polymorphism (T-C) leading to V736A change in the primary structure of MT2 may cause fine alterations in the tertiary conformation of the catalytic domain, leading to changes in the thermodynamic stability of the complex it forms with HJV as observed with the in silico study. Accordingly, the MT2 736 V variant which is encoded by the rs855791 T allele may not be able to catalyze the cleavage of HJV efficiently enough due to relatively low thermodynamic stability causing the BMP pathway to continue, despite low serum iron levels. This might disrupt the negative regulation of HAMP gene and could result in relatively high hepcidin levels. Consequently, dietary iron absorption and release of iron from macrophages may be hampered leading to low iron levels in blood circulation . Continuation of this cycle for prolonged periods may lead to iron deficiency . The presence of TT genotype could augment this effect as MT2 enzymes encoded by both copies of the TMPRSS6 gene are altered.
On the other hand, TC genotype has not made a significant effect on iron levels within the study population. This suggests that having a single C allele could cover up for the altered MT2 enzymes encoded by the rs855791T allele among heterozygotes, establishing the normal iron homeostasis. As such, those with TC would carry sufficient amount of iron in their serum similar to those who carry CC genotype, while TT homozygotes are prone to become iron deficient.
Our study is in agreement with two previous studies conducted among Italian  and Taiwanese  populations, which reported a similar distribution of serum iron levels with the two rs855791 variants. In the latter study, the CC genotype was reported to be associated with a protective role for IDA with a OR of 0.4 (95% CI, 0.17–0.95, p = 0.04) supporting the observations of the present investigation. Interestingly, in all these populations (Sri Lankans, Italians, Taiwanese) the rs855791 T allele that encodes MT2 736 V was present at a higher frequency than its counterpart, despite its negative impact on iron homeostasis. This along with its evolutionary conserved nature when analyzed in comparison with other species who are closely related to humans  further suggests that MT2 736 V variant, which leads to increased hepcidin production and inhibition of iron absorption leading to low iron levels in the circulation, is a recent evolutionary change. Such switching of SNP alleles could have arisen due to systemic inflammation conditions which might have been prevalent among the populations over generations due to numerous environmental stresses. For example, high hepcidin levels which leads to low serum iron in turn have shown to be beneficial in counteracting malaria [41,42,43].
On the other hand, the low dietary iron intake among the current study population may have contributed to the high prevalence of iron deficiency. The lack of nutritional awareness as well as financial constrains might have played a role in causing this situation. It is also possible that the inadequate iron intake in these pregnant women has inflated the intensity of the genetic association observed between rs855791 and serum iron levels. Recruiting subjects with adequate dietary iron intake would help to remove this confounding factor and to reveal a more accurate picture of serum iron modulation by rs855791. Limited sample size prevented us from exploring this possibility during the analysis. However, it should also be noted that the dietary iron consumption data of this study suffers from recall bias which is a well-known drawback in recall food intake data. In addition, causes such as parasitic infestations and viral infections leading to underlying chronic inflammation, which are known to prevail in tropical countries, may have contributed to aggravate the effect of genetic modulation on iron metabolism. Notwithstanding these limitations, the current study highlights the requirement of stringent public health interventions to prevent iron deficiency among pregnant women in Sri Lanka.
Despite this significant association shown by rs855791 with serum iron levels, the genotype frequencies of the SNP locus were consistent with the Hardy–Weinberg equilibrium (HWE) expectations indicating the absence of significant evolutionary forces acting upon it. This observation agrees with the hypothesis put forward by Nai et al.  suggesting that the association of this SNP with iron deficiency is only a recent evolutionary change as discussed above. Otherwise, it is unlikely for a functionally significant polymorphism to remain neutral over an evolutionary time scale.
Bioinformatic analysis of rs855791 was conducted to find out the molecular mechanisms of iron regulation via BMP-SMAD signaling pathway. As indicated by the validation criteria, all the generated models were reliable. These predicted models would be useful in the in silico screening of potential drug targets for ID. The natural behavior of amino acids in the secondary structure and the complete tertiary structure has the ability to change the protein stability . Nevertheless, which of these would affect more on the protein stability in most cases is unclear. Though the change between valine and alanine is only a CH2CH3 group, with its location they can influence the protein stability. A well-known example that demonstrates the effect of subtle changes in the primary structure of a protein on its conformation and function is found in the case of sickle cell anemia (the amino acid change in glutamic acid to valine causes the disease) . The secondary structural elements of a protein may evolve at different rates. According to Siltberg-Liberies et al. , the evolution rate of beta sheets is slower than helical regions and random coils. Some of these could be evolutionarily neutral, while some others can have strong positive or negative selection. In the present scenario, the variant region is in a beta pleated sheet surrounded by several random coils. CH2CH3 group of valine acquires more three-dimensional space when compared with that of alanine. Thus, with alanine it would be easier to coil since the inter-spatial space is low, which makes the protein more stable.
When the obtained interface energy values of the complexes were compared, MT2 736 A variant—HJV, is more thermodynamically stable than the MT2 736 V variant—HJV complex. With a higher stability, MT2 736 A variant is likely to cleave HJV molecule more efficiently with a better protease activity compared to its counterpart, the MT2 736 V variant. This might result in a more efficient inhibition of BMP-HJV pathway, negatively regulating the HAMP gene transcription. This will lead to lower production of hepcidin levels resulting in high iron levels in blood circulation.
In this post genomic era, identifying those markers that confer genetic predisposition to complex diseases is of much interest. However, due to the differences in genetic makeup among different ethnicities and the complex interactions they have with their environment, not all such markers could be equally effective to all populations . Our results, though only preliminary considering the number of samples analyzed, indicate that SNP rs855791 can be successfully used as a molecular marker to screen iron deficiency risk among Sri Lankan women especially of the susceptible populations such as women of childbearing age, pregnant women, adolescent girls and the young child. Such early detection and correction of iron deficiency could be one of the first steps of ensuring a healthy future generation in those developing nations including Sri Lanka. In addition, since biochemical markers such as ferritin levels can be masked by inflammation, use of molecular makers would be advantageous .
Furthermore, this knowledge on the molecular mechanism of iron deficiency may also be helpful for therapeutic interventions. Although as pointed out by Galesloot et al. , rs855791 may also employ a hepcidin-independent mechanism to regulate iron metabolism, the iron deficiency associated with rs855791T allele is at least partly caused by hepcidin deregulation as suggested by our results and many previous studies [19, 20, 47]. Hence, in addition to treating with elemental iron supplements, alternative treatment methods such as hepcidin blockers (small interfering RNA/ antibodies) or alternative supplements using heme iron may be used to ensure iron absorption . This is especially valid for women with inflammation as previous results from the same cohort highlight the ineffectiveness of elemental iron supplements in increasing serum ferritin in pregnant women who had inflammation .
However, it is important to confirm the observed risk of iron deficiency modulated by rs855791 through a larger case control study excluding the confounding factors such as low dietary iron intake, parity and diverse ethnicity that are commonly associated with iron deficiency . Measuring the hepcidin levels in the subjects would also aid in determining the molecular mechanism of iron regulation via rs855791 polymorphism.