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ORIGINAL ARTICLE
Year : 2022  |  Volume : 35  |  Issue : 3  |  Page : 1314-1317

Effect of TMPRSS6 rs855791 (T>C) polymorphism on pathophysiology of iron-deficiency anemia


1 Department of Pediatrics, Shyam Shah Medical College, Rewa, Madhya Pradesh, India
2 Deptartment of Zoology, Govt. Girl PG College, Rewa, Madhya Pradesh, India
3 Department of Anesthesiology, Shyam Shah Medical College, Rewa, Madhya Pradesh, India
4 Deptartment of Biotechnology, Guru Ghasidas University, Bilaspur, Chhattisgarh, India
5 Department of Multidisciplinary Research Unit, Shyam Shah Medical College, Rewa, Madhya Pradesh, India

Date of Submission28-Dec-2021
Date of Decision09-Apr-2022
Date of Acceptance10-Apr-2022
Date of Web Publication29-Oct-2022

Correspondence Address:
Sanjay Kumar Pandey
Multidisciplinary Research Unit, Shyam Shah Medical College, Rewa, Madhya Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mmj.mmj_316_21

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  Abstract 


Background
The TMPRSS6 (Transmembrane Serine Protease 6) gene is found on chromosome 22. TMPRSS6 is a protein-coding gene. Iron absorption and metabolism in the body is controlled by matripase-2 enzyme, which is encoded by TMPRSS6 gene.
Objectives
TMPRSS6 rs855791 polymorphism was investigated for its effect on serum iron levels of patients with iron-deficiency anemia.
Patients and methods
A tertiary referral hospital served as the study setting. The complete blood count was analyzed using an automated five-part hemtoanalyzer. Enzyme-linked immunosorbent assay (ELISA) was used to analyze ferritin in serum. Restriction fragment length polymorphism PCR-RFLP was used to genotype TMPRSS6rs855791 C>T iron assimilator gene using StuI restriction enzyme.
Results
There were 21 heterozygous patients and four homozygous patients. TMPRSS6 (rs855791C>T) mutants were found to present 14 heterozygotes, whereas no homozygotes were present in controls. Serum ferritin and hemoglobin levels were also elevated in mutants with this mutation. C-reactive protein (CRP) levels and Erythrocyte sedimentation rate (ESR) levels were decreased in mutant genotypes. These parameters were statistically significant. The total iron-binding capacity level and % transferrin saturation levels were significantly higher in TMPRSS6 (rs855791C>T) genotypes, and the P values were statistically significant. TMPRSS6 (rs855791C>T) mutation is associated with less severe iron-deficiency anemia symptoms.
Conclusion
As the results of our study indicate, mutations protect against iron-deficiency anemia and confer selection advantage to heterozygous carriers.

Keywords: iron deficiency anemia, rs855791, TMPRSS6


How to cite this article:
Pandey S, Kunder S, Singh J, Bajaj N, Dwivedi S, Baghel VS, Pandey D, Pandey SK. Effect of TMPRSS6 rs855791 (T>C) polymorphism on pathophysiology of iron-deficiency anemia. Menoufia Med J 2022;35:1314-7

How to cite this URL:
Pandey S, Kunder S, Singh J, Bajaj N, Dwivedi S, Baghel VS, Pandey D, Pandey SK. Effect of TMPRSS6 rs855791 (T>C) polymorphism on pathophysiology of iron-deficiency anemia. Menoufia Med J [serial online] 2022 [cited 2024 Mar 29];35:1314-7. Available from: http://www.mmj.eg.net/text.asp?2022/35/3/1314/359498




  Introduction Top


Protection against iron deficiency and its morbid consequences may have selective advantage for heterozygous carriers of the mutation in patients. Hence, we hypothesized that the presence of TMPRSS6 mutations may prevent or ameliorate iron deficiency in patients. Among many, iron deficiency is one of the most frequently occurring nutritional disorders, globally affecting a large portion of population[1]. There can be many reasons for the iron deficiency in the people, including diet deficient in iron, metabolic disorders, and genetic factors. Iron absorption and metabolism in the body is regulated by hepcidin, which itself is tightly regulated by a variety of other factors[2]. Among these, matripase-2, a serine protease transcribed by the TMPRSS6 gene, is the major one, and it is known to downregulate the expression of hepcidin. Matripase-2 achieves this downregulation of transcription by cleaving the hemojuvelin bound to the membrane[2],[3],[4],[5]. The downregulation of hepcidin has been linked with iron overload in animal models and humans, whereas its upregulation has been linked with iron-deficiency anemia. Complete loss of TMPRSS6 has been linked with a rare anemia, iron-refractory iron-deficiency anemia. The patients with iron-refractory iron-deficiency anemia remain unresponsive toward orally administered iron. They have very high hepcidin level and hence are unable to absorb iron from intestine[4],[6]. Various recent genome-wise studies have reported multiple single nucleotide polymorphisms (SNPs) of TMPRSS6 gene, which are known to directly influence the hematological phenotypes and red blood cell (RBC) indices. Among all, rs855791 (2321 C>T) has shown the sturdiest relationship with iron parameters and RBC indices[7],[8]. This SNP is found in the functional region of TMPRSS6 gene, and this alanine to valine nonsynonymous change leads to the reduced ability of inhibiting hepcidin transcription. Transmembrane serine protease is a product of the TMPRSS6 gene that interferes with hepcidin production and has led to the discovery of a severe form of iron-deficiency anemia owing to mutations in both paternal and maternal TMPRSS6 genes that is refractory to iron treatment. TMPRSS6 or matriptase-2 is a member of a family of cell surface proteolytic enzymes defined as type II transmembrane serine proteases and is a physiological suppressor of hepcidin. The available evidence suggests that in the absence of TMPRSS6, increased hepcidin concentrations degrade intestinal ferroprotein and interfere with normal iron absorption[3],[9]. In fact, a splicing error in TMPRSS6 has been detected in Mask mice, which have a recessive chemically, induced phenotype characterized by progressive loss of body hair and severe iron deficiency owing to reduced absorption of iron from the gastrointestinal tract. Mask mice produce a truncated copy of the TMPRSS6 protein lacking the serine protease domain and express inappropriately high levels of hepcidin. That these high levels can be responsible for iron deficiency and severe microcytic anemia was previously observed in transgenic mice overexpressing hepcidin[10]. In addition, another recent study indicates that iron-deficiency anemia refractory to oral iron therapy can be caused by various germline mutations in TMPRSS6. Proteolytic events at the cell surface regulate many important cellular processes[4],[11]. Matriptase-2 appears to play a crucial role in body iron homeostasis, and polymorphisms or mutations of this gene may partly explain the variability in iron absorption in humans. In the future, TMPRSS6 might also be a target for manipulation of hepcidin production in disorders of iron metabolism. This case–control study was conducted to investigate the effect of TMPRSS6 rs855791 polymorphism in the pathophysiology of children with iron-deficiency anemia.


  Patients and methods Top


A total of 140 patients with iron-deficiency anemia were included in this study. Of them, 75 were male (mean age = 11.2 ± 5.3 years) and 65 were female (mean age = 12.1 ± 4.6) patients. Organizing medical camps, attending patients to the hospital, and personal contacts were used to recruit patients. The total number of healthy age-matched and sex-matched control samples was 140 (75 males and 65 females, with a mean age of 10.3 ± 4.7 and 11.2 ± 5.1 years, respectively). The study was approved by the institutional ethical committee of Shyam Shah Medical College Rewa, Madhya Pradesh India. A detailed in-person questionnaire was administered, and a specially designed form was used to collect demographic data. Upon receiving informed and written consent from the participants or their parents/guardians, all study participants were enrolled. All patients except those experiencing iron deficiency were excluded from this study. Approximately 5 ml of venous blood was collected from the participants and tests for complete blood count and serum iron were done. An automated 5-part hemtoanalyzer (SYSMEX XS-800i, Kobe, Japan) was used to analyze complete blood count. The normal cutoff values were defined as follows: white blood cells, 3.5–11.5 ths/μl; RBCs, 2.5–5.5 million/μl; hemoglobin, 10.9–15.0 g/dl; hematocrit, 26.0–50.0%; mean corpuscular volume, 80–100 fl.; mean corpuscular hemoglobin, 26.0–38.0 pg; mean corpuscular hemoglobin concentration, 31.0–37.0 g/dl; and platelet count, 150–450 ths/μl. For the determination of iron deficiency in the patients' serum, ferritin analysis was done by the ELISA method using human FTL IMMUNO TAG kit (G-Biosciences, St Louis, USA). Microlab's ELISA Reader (Microlisa PLUS; Microlab, Ahmedabad, Gujarat, India) and washer (WRX-806PLUS) were used. The values suggestive of iron deficiency were defined as follows: serum ferritin, less than 12 μg/l; total iron-binding capacity, 300–400 μg/dl (>400 μg/dl in iron-deficiency anemia); % transferrin saturation, 20–55%; CRP, 0.02–14.4 mg/l; and ESR, less than or equal to 10 mm/h. The isolation of total genomic DNA was done from peripheral blood leukocytes using Genomic DNA mini-Gene aid kit as per manufacturer's protocol. After the isolation DNA was quantified using Nano drop analytical software system, the genotyping for TMPRSS6rs855791 C>T iron assimilator gene was done by PCR (iGene Labserve, New Delhi, India) and polymorphism was characterized by PCR-RFLP method using StuI restriction enzyme (New England Biolabs (NEB), Ipswich, Massachusetts, United States of Ammerica) according to the published literature[1],[2],[12]. The sequences of primers (BR Biochem, New Delhi, India) for PCR amplification were 5′-TAGAGAACAGGGGCTCCAGG-3′-forward and 5′-ATGTGGGCAGCATCCTTTC-3′-reverse. TMPRSS6 (rs855791C>T) primers was designed and characterized using the PCR-RFLP method. DNA of TMPRSS6 (rs855791C>T) resulted in a PCR product of 249 bp when visualized in agarose gel. StuI restriction enzyme was used for TMPRSS6 (rs855791C>T) SNP. Digested product was checked in 3% ethidium bromide containing agarose gel. After restrictive digestion of TMPRSS6 (rs855791C>T) PCR product, various fragmented products were seen in agarose gel, which were tracked through double-digested bromophenol blue dye. Three genotypic patterns were seen in the studied patients. A 249-bp product size represents wild-type CC homozygous, a 249- and 125-bp product size represents heterozygous CT conditions, whereas 125 bp product size represents mutant homozygous condition. Data were entered in Microsoft Excel 2007 (USA), and all statistical analyses were performed with GraphPad Prism software version 4.00 (San Diego, California, USA). Descriptive characteristics (mean and SD) and percentage were performed for each parameter separately. Independent t test was used for proportions and mean comparisons between case and control groups, respectively. P value less than 0.05 was considered statistically significant.


  Results Top


The present study collected data from 140 patients with IDA and 140 healthy age-matched and sex-matched controls. Patients and controls were evaluated for frequency of TMPRSS6 (rs855791C>T) SNP. In mutant and wild-type genotypes of cases, clinical symptoms were recorded. It was reported that 84% of children with mutant IDA were weak, 84% easy fatigable, 50% were breathless, 28% were tachycardic, 32% had systolic heart murmur, 36% had atrophic glossitis, 25% had angular stomatitis, and 64% had pica, whereas 94.78% were weak, 90.04% were easy fatigable, 57.06% were breathless, 56.52% had tachycardia, 57.39% had systolic heart murmurs, 36.52% had atrophic glossitis, 62.6% had angular stomatitis, and 73.04% had picas in nonmutant IDA. children. Among children with IDA, the wild-type genotype was more likely to exhibit clinical symptoms. Comparative clinical frequency of TMPRSS6 (rs855791C>T) mutant versus nonmutant is given in [Figure 1]. A total of 21 of the patients were heterozygous and four were homozygous, whereas the controls had 14 heterozygous genotype cases, but no homozygous cases of TMPRSS6 (rs855791C>T) genotype. The severity of symptoms was worsened among nonmutants. The mean hemoglobin values for the mutants (n = 25) and nonmutants (n = 115) were 8.3 ± 1.3 and 7.4 ± 1.2 g/dl, respectively (P = 0.0001). The mean values of serum ferritin in mutants and nonmutants were 11. 4 ± 2.3 and 9.3 ± 2.4 μg/l, respectively (P = 0.0001). The mean values of ESR were 18 ± 4 and 25 ± 3 mm/h in the mutant and nonmutant, respectively (P = 0.0001). The mean value of CRP in mutant was 0.9 ± 0.5 and 1.3 ± 0.4 mg/l among the nonmutant (P = 0.0001). Detailed comparative parameters of TMPRSS6 (rs855791C>T) mutant versus nonmutant are given in [Table 1].
Figure 1: Comparative clinical frequency of TMPRSS6 (rs855791C>T) mutant versus wild-type genotype of cases.

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Table 1: Comparative parameters of TMPRSS6 (rs855791C>T) mutant versus nonmutant

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  Discussion Top


In a study by Gan et al.[13], we also observed elevated serum ferritin and hemoglobin levels in TMPRSS6 (rs855791C>T) mutants. The mutant genotype has decreased CRP and ESR levels. These parameters were statistically significant. In the TMPRSS6 genotype (rs855791C>T), the total iron-binding capacity level and transferrin saturation level were increased, and the P values were significant. Symptoms of iron-deficiency anaemia are less frequent in individuals carrying the rs855791C>T mutation in TMPRSS6. Previously, Nai et al.[14] reported that rs855791C>T is strongly associated with serum iron, transferrin saturation, and hematological parameters; it was also shown to increase iron parameters in patients by inhibiting hepcidin. Pei et al.[1] further confirmed this role of rs855791C>T, reporting that it protects women against iron-deficiency anemia. The results of our study demonstrate that the heterozygous (TC) genotype confers protection against iron-deficiency anemia, in agreement with previous studies[12],[15],[16]. The findings of our study contradict those of Kumar et al.[17] and Macdougall et al.[18], who reported that CC genotype is protective in IDA, whereas TT genotype is pathological. Later studies by Gonçalves et al.[19] reported that patients with IDA were less likely to have CC genotypes. Variations in the study may be attributable to a difference in the study population and ethnicity. These data demonstrated that TMPRSS6 (rs855791C>T) genotypes serve as effective predictors of IDA. Therefore, patients with IDA should be tested for TMPRSS6 (rs855791C>T) genotype. This can help guide baseline investigations and treatment decisions.


  Conclusion Top


The available evidence suggests that in the absence of TMPRSS6, increased hepcidin concentrations degrade intestinal ferroprotein and interfere with normal iron absorption. SNPs of TMPRSS6 gene are known to directly influence the hematological phenotypes and RBC indices. Our data strongly suggest a role of the TMPRSS6 (rs855791C>T) mutation in preventing iron deficiency in Asian Indian population. Protection against iron deficiency and its morbid consequences has conferred a selective advantage for heterozygous carriers of the mutation.

Acknowledgements

Contributors: S.K.P., N.B., and S.K.: concept and design of the study, analysis of data, and drafting of the manuscript; S.P., D.P., and V.S.B.: collected the data and helped in data analysis; and S.D.: supervised and provided facility for research work.

Financial support and sponsorship

This study is funded by Indian Council of Medical Research, New Delhi.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Pei SN, Ma MC, You HL, Fu HC, Kuo CY, Rau KM, et al. TMPRSS6 rs855791 polymorphism influences the susceptibility to iron deficiency anemia in women at reproductive age. Int J Med Sci 2014; 11:614–619.  Back to cited text no. 1
    
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Nalado AM, Dickens C, Dix-Peek T, Mahlangu JN, Olorunfemi G, Paget G, et al. TMPRSS6 rs855791 polymorphism and susceptibility to iron deficiency anaemia in non-dialysis chronic kidney disease patients in South Africa. Int J Mol Epidemiol Genet 2019; 10:1–9.  Back to cited text no. 12
    
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Gan W, Guan Y, Wu Q, Peng A, Zhu J, Ling L, et al. Association of TMPRSS6 polymorphisms with ferritin, hemoglobin, and type 2 diabetes risk in a Chinese Han population. Am J Clin Nutr 2012; 95:626–632.  Back to cited text no. 13
    
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Nai A, Pagani A, Silvestri L, Campostrini N, Corbella M, Girelli D, et al. TMPRSS6 rs855791 modulates hepcidin transcription in vitro and serum hepcidin levels in normal individuals. Blood 2011; 118:4459–4462.  Back to cited text no. 14
    
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Gonçalves L, Nobre Jesus G, Afonso C, Vieira A, Maia R, Correia L, et al. The role of TMPRSS6 gene variants in different types of iron deficiency anaemia-from the rare severe hereditary IRIDA to the common mild acquired IDA. Reunião Científica do Anemia Working Group Portugal 28-29 novembro 2014.  Back to cited text no. 19
    


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