The role of TMPRSS6 polymorphisms in iron deficiency anemia partially responsive to oral iron treatment Poggiali, Erika1; Andreozzi, Fabio1; Nava, Isabella1; Consonni, Dario2; Graziadei, Giovanna1; Cappellini, Maria Domenica1 1
IRCCS Fondazione Ca’ Granda Ospedale Maggiore Policlinico, Università degli Studi di Milano,
Dipartimento di Medicina Interna 2
Consonni, Dario; IRCCS Fondazione Ca’ Granda Ospedale Maggiore Policlinico, Unità di
Epidemiologia, Dipartimento di Medicina preventiva
ABSTRACT Iron Refractory Iron Deficiency Anemia (IRIDA) is a rare hereditary disease caused by mutations in TMPRSS6 gene encoding Matriptase-2, a negative regulator of hepcidin transcription. Up to now, 53 IRIDA patients from 35 families with different ethnic origins have been reported and 41 TMPRSS6 mutations have been identified. TMPRSS6 polymorphisms are more frequent than mutations, and have been associated with variation in iron and hematologic parameters. Our study evaluated their presence in 113 subjects with iron deficiency anemia (IDA) partially responsive to oral iron therapy and in 50 healthy blood donors. Thalassemic trait was diagnosed in 38 patients. Sequencing analysis of TMPRSS6 gene revealed that the frequency of several polymorphisms was markedly
different
between
IDA
subjects
and
controls.
In
particular,
the
V736A TMPRSS6 polymorphism was associated to moderately lower hemoglobin, mean corpuscular volume and mean corpuscular hemoglobin levels, and in thalassemia carriers with marked anemia and microcytosis. A new variant - H448R- and two uncommon polymorphisms A719T and V795I - were also identified. These results indicate that TMPRSS6 polymorphisms are more frequent in subjects with persistent IDA than in healthy controls, and in thalassemia carriers V736A variant may account for lower hemoglobin and MCV levels. Further studies in larger court of patients are necessary to identify potential haplotypes and polymorphisms responsible for low response to oral iron treatment and may be useful for planning a correct iron supplementation. Keywords: Anemia- Nutritional, iron, cobalamin, folate, Thalassemia, Iron- absorption and metabolism, Iron, Hemoglobin
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1002/ajh.23929 This article is protected by copyright. All rights reserved.
INTRODUCTION Iron deficiency anemia (IDA) is the most common worldwide anemia with important clinical consequences [1], caused by inadequate iron availability for hemoglobin production due to different factors. The lack of dietary iron, the insufficient uptake of iron and chronic bleeding are the most frequent causes. Helicobacter Pylori should be always considered as possible cause of IDA and be investigated in subjects with persistent anemia [2]. Recently, genetic factors have been identified that may play an important role in the pathogenesis of IDA. Iron is essential for life and it is an important cofactor for several enzymatic reactions involved in human physiology [3], although if in excess, iron can react with oxygen species to form active free radicals that damage macromolecules and cellular organelles [4]. For these reasons, in the human body the iron homeostasis is tightly regulated in order to avoid both deficiency and toxicity due to iron excess. Hepcidin is the key hormone regulator of systemic iron homeostasis. The role of hepcidin is to control the surface expression of the iron exporter ferroportin on duodenal enterocytes and macrophages, modulating iron absorption and recycling [5]. Inactivation of hepcidin causes severe iron overload, while hepcidin over expression causes anemia [6]. Several factors contribute to the regulation of hepcidin synthesis: the bone morphogenetic proteins (BMPs) - hemojuvelin (HJV) complex [7] and the interleukin-6 - signal transducer and activator of transcription (STAT3) pathway [6] increase hepcidin synthesis, while matriptase-2 down regulates hepcidin synthesis. Matriptase-2 (MT-2) is a type II transmembrane serine protease encoded by the transmembrane protease, serine 6 (TMPRSS6) gene, mapped on 22q12-13 [8, 9], and mainly expressed in the liver [10]. In vitro MT-2 cleaves the BMP-coreceptor HJV [11], down regulating hepcidin expression [12]. The “mask” mouse, a model animal with deleted serine protease domain, showed microcytic anemia consequent to elevated hepcidin levels [8]. In humans TMPRSS6 inactivation causes iron-refractory iron-deficiency anemia (IRIDA, OMIM #206200, ORPHA209981). IRIDA is a rare autosomal recessive disorder hallmarked by hypochromic microcytic anemia, with low plasma iron, low transferrin saturation, and ferritin usually within the normal range. Moreover, hepcidin could be normal/high, and the anemia is unresponsive to oral iron treatment and only partially responsive to parenteral iron supplementation [9,13-17]. The prevalence of IRIDA is estimated to be less than 1/1000000. So far 53 cases from 35 families with different ethnic origin have been reported, and 43 mutations in the TMPRSS6 gene have been identified [18,19]. Although mutations are extremely rare, recent insights have revealed that highly TMPRSS6 polymorphisms may influence iron absorption [20]. Single Nucleotide Polymorphisms 2 This article is protected by copyright. All rights reserved.
(SNPs) refer to single base pair positions in DNA at which different alleles exist. Genome-wide association studies identified common genetic polymorphisms in TMPRSS6 associated with iron status, hemoglobin level and erythrocyte volume [21-28]. The strongest association was with rs855791, consistent with the hypothesis that rs855791 influences hepcidin-regulated iron homeostasis [21;27]. Beutler et al. investigated the role of polymorphisms of TMPRSS6 genes in iron deficient adults [29], based on the observation that polymorphisms of TMPRSS6 might be risk factors for iron deficiency anemia [8]. These evidences highlight the role of MT-2 in the control of iron metabolism and erythrocyte parameters. These evidences highlight the role of MT-2 in the control of iron metabolism and erythrocyte parameters. Recently, reported cases underline that particular polymorphisms, in combination with a heterozygous TMPRSS6 mutation, can account for overt clinical presentation [26-28;30]. The SNP rs855791 is the polymorphism most associated with iron and hematologic parameters. According to dbSNP genetic variations archive (NCBI), rs855791 identifies T>C substitution in position 2207 (correspondent to a complementary A>G change), resulting the missense valine (V) to alanine (A) change in position 736 (p.V736A variant), nearby catalytic and binding sites of MT-2. The human database indicates V at the 736 position as the “wild type” amino acid of MT-2; A is evolutionary conserved in other species, suggesting it is the ancestral amino acid and that V in position 736 is a recent evolutionary change [31]. In this study we investigated the presence and the role of the TMPRSS6 polymorphisms in patients with IDA, referred to our outpatients service because only partially responsive to oral iron therapy. Fifty healthy blood donors were used as control group. TMPRSS6 sequencing has been performed with these aims: a) to identify TMPRSS6 polymorphisms in enrolled population and in healthy control group; b) to identify cases of pure IRIDA.
METHODS Study subjects Between January 2009 and December 2013, 113 subjects (12 males, 101 females) with mean age 36 ± 13 years (range 4-74 years) were referred to the Rare Diseases Centre of “Fondazione IRCCS Ca’ Granda, Ospedale Maggiore Policlinico di Milano” for persistent IDA poorly responsive to oral iron. All the subjects were previously treated with oral iron treatment by their general practitioners or other hematologists with incomplete response even in presence of good patient’s compliance and proper treatment dose and duration. Most of the patients reported a mild degree of iron deficiency anemia during childhood or adolescence, treated with repeated cycles of oral iron therapy, but 3 This article is protected by copyright. All rights reserved.
always with partial benefits. All the patients reported normal growth and a good quality of life. In all the cases we investigated celiac disease, gastrointestinal bleeding and Helicobacter Pylori (HP) infection (Figure 1). Hematologic parameters, iron status, inflammatory markers and thyroid function were tested. Fifty healthy donors ethnic-matched to the subjects enrolled in the study, were used as control groups (Table I). Sequence variation in TMPRSS6 gene was evaluated by PCR and direct sequencing in genomic DNA isolated from peripheral lymphomonocytes. Direct sequencing was performed using a fluorescence-tagged dideoxy chain terminator method in an ABI BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA, USA), according to the manufacturer’s instructions. Thalassemia trait was suspected and investigated in 38 out of 113 patients (34%; 3 males, 35 females) using HPLC and molecular analysis of globin genes. Beta globin chain gene has been investigated for the common Mediterranean mutations, including cod. 39, cod. 6, IVS1-1, IVS2-1 (β°), and -101, -87, IVS1-6, IVS1-110, IVS2-745 (β+), by DGGE (Denaturing Gradient Gel Electroforesis) after PCR (polymerase chain reaction) DNA amplification. The analysis of α-globin genes has been performed in selected patients. The study has been conducted according to Helsinki Declaration. All the recruited patients have given their signed informed consent. For statistical analysis, multiple linear regression models adjusted for sex and age (continuous) was used (Armitage P, Berry G, Matthews JNS. Statistical methods in medical research; Fourth Edition, Oxford, Blackwell Science 2002) as well as multiple logistic regression models were used (Clayton D, Hills M. Statistical models in epidemiology, Oxford; New York: Oxford University Press, 1993). We calculated sex- and age-adjusted odds ratios (OR) and 95% confidence intervals (95% CI) of being a case for V736A homozygosis, heterozygosis and wild type. When OR was not estimable because of zero controls, we reported the p-value from chi-square test. Analyses were performed with Stata 13 (StataCorp. 2013. Stata: Release 13. Statistical Software. College Station, TX: StataCorp LP).
RESULTS The hematologic characteristics and iron status of patients and controls are reported in Table I. Considering the common TMPRSS6 polymorphisms, we found that the frequencies of V736A, SNP-120, SNP-113, F5F, P33P, K253E, S361S, Y418Y, D521D, Δ15accc and Y739Y were markedly different between patients and controls (Figure 2, Table II). Homozigosity for V736A was detected in 40 patients (35.5%), whereas it was never detected in healthy controls; forty-eight (42.5%) patients resulted heterozygotes, as reported in Figure 2.
4 This article is protected by copyright. All rights reserved.
Hematologic parameters in enrolled population according to genotype (homozygotes, heterozygotes and wild type for V736A) are reported in Table III. Considering together patients and controls, we found that the presence of the polymorphism V736A both in heterozygosis and in homozygosis is associated to lower hemoglobin levels (respectively, -0.6 gr/dL, p=0.05; and -0.9 gr/dL, p=0.01) as well as a statistically significant variation in MCV and MCH values was observed (Table IV). Agreement percentage between V736A and other polymorphisms in whole population are reported in Table V. The relationship with the common polymorphisms D521D and Y739Y are shown in Tables VI and VII. A new variant - H448R - was identified in a patient with IDA requiring parenteral iron supplementation. Two uncommon polymorphisms, A719T and V795I (estimated frequency
IRCCS Fondazione Ca’ Granda Ospedale Maggiore Policlinico, Università degli Studi di Milano,
Dipartimento di Medicina Interna 2
Consonni, Dario; IRCCS Fondazione Ca’ Granda Ospedale Maggiore Policlinico, Unità di
Epidemiologia, Dipartimento di Medicina preventiva
ABSTRACT Iron Refractory Iron Deficiency Anemia (IRIDA) is a rare hereditary disease caused by mutations in TMPRSS6 gene encoding Matriptase-2, a negative regulator of hepcidin transcription. Up to now, 53 IRIDA patients from 35 families with different ethnic origins have been reported and 41 TMPRSS6 mutations have been identified. TMPRSS6 polymorphisms are more frequent than mutations, and have been associated with variation in iron and hematologic parameters. Our study evaluated their presence in 113 subjects with iron deficiency anemia (IDA) partially responsive to oral iron therapy and in 50 healthy blood donors. Thalassemic trait was diagnosed in 38 patients. Sequencing analysis of TMPRSS6 gene revealed that the frequency of several polymorphisms was markedly
different
between
IDA
subjects
and
controls.
In
particular,
the
V736A TMPRSS6 polymorphism was associated to moderately lower hemoglobin, mean corpuscular volume and mean corpuscular hemoglobin levels, and in thalassemia carriers with marked anemia and microcytosis. A new variant - H448R- and two uncommon polymorphisms A719T and V795I - were also identified. These results indicate that TMPRSS6 polymorphisms are more frequent in subjects with persistent IDA than in healthy controls, and in thalassemia carriers V736A variant may account for lower hemoglobin and MCV levels. Further studies in larger court of patients are necessary to identify potential haplotypes and polymorphisms responsible for low response to oral iron treatment and may be useful for planning a correct iron supplementation. Keywords: Anemia- Nutritional, iron, cobalamin, folate, Thalassemia, Iron- absorption and metabolism, Iron, Hemoglobin
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1002/ajh.23929 This article is protected by copyright. All rights reserved.
INTRODUCTION Iron deficiency anemia (IDA) is the most common worldwide anemia with important clinical consequences [1], caused by inadequate iron availability for hemoglobin production due to different factors. The lack of dietary iron, the insufficient uptake of iron and chronic bleeding are the most frequent causes. Helicobacter Pylori should be always considered as possible cause of IDA and be investigated in subjects with persistent anemia [2]. Recently, genetic factors have been identified that may play an important role in the pathogenesis of IDA. Iron is essential for life and it is an important cofactor for several enzymatic reactions involved in human physiology [3], although if in excess, iron can react with oxygen species to form active free radicals that damage macromolecules and cellular organelles [4]. For these reasons, in the human body the iron homeostasis is tightly regulated in order to avoid both deficiency and toxicity due to iron excess. Hepcidin is the key hormone regulator of systemic iron homeostasis. The role of hepcidin is to control the surface expression of the iron exporter ferroportin on duodenal enterocytes and macrophages, modulating iron absorption and recycling [5]. Inactivation of hepcidin causes severe iron overload, while hepcidin over expression causes anemia [6]. Several factors contribute to the regulation of hepcidin synthesis: the bone morphogenetic proteins (BMPs) - hemojuvelin (HJV) complex [7] and the interleukin-6 - signal transducer and activator of transcription (STAT3) pathway [6] increase hepcidin synthesis, while matriptase-2 down regulates hepcidin synthesis. Matriptase-2 (MT-2) is a type II transmembrane serine protease encoded by the transmembrane protease, serine 6 (TMPRSS6) gene, mapped on 22q12-13 [8, 9], and mainly expressed in the liver [10]. In vitro MT-2 cleaves the BMP-coreceptor HJV [11], down regulating hepcidin expression [12]. The “mask” mouse, a model animal with deleted serine protease domain, showed microcytic anemia consequent to elevated hepcidin levels [8]. In humans TMPRSS6 inactivation causes iron-refractory iron-deficiency anemia (IRIDA, OMIM #206200, ORPHA209981). IRIDA is a rare autosomal recessive disorder hallmarked by hypochromic microcytic anemia, with low plasma iron, low transferrin saturation, and ferritin usually within the normal range. Moreover, hepcidin could be normal/high, and the anemia is unresponsive to oral iron treatment and only partially responsive to parenteral iron supplementation [9,13-17]. The prevalence of IRIDA is estimated to be less than 1/1000000. So far 53 cases from 35 families with different ethnic origin have been reported, and 43 mutations in the TMPRSS6 gene have been identified [18,19]. Although mutations are extremely rare, recent insights have revealed that highly TMPRSS6 polymorphisms may influence iron absorption [20]. Single Nucleotide Polymorphisms 2 This article is protected by copyright. All rights reserved.
(SNPs) refer to single base pair positions in DNA at which different alleles exist. Genome-wide association studies identified common genetic polymorphisms in TMPRSS6 associated with iron status, hemoglobin level and erythrocyte volume [21-28]. The strongest association was with rs855791, consistent with the hypothesis that rs855791 influences hepcidin-regulated iron homeostasis [21;27]. Beutler et al. investigated the role of polymorphisms of TMPRSS6 genes in iron deficient adults [29], based on the observation that polymorphisms of TMPRSS6 might be risk factors for iron deficiency anemia [8]. These evidences highlight the role of MT-2 in the control of iron metabolism and erythrocyte parameters. These evidences highlight the role of MT-2 in the control of iron metabolism and erythrocyte parameters. Recently, reported cases underline that particular polymorphisms, in combination with a heterozygous TMPRSS6 mutation, can account for overt clinical presentation [26-28;30]. The SNP rs855791 is the polymorphism most associated with iron and hematologic parameters. According to dbSNP genetic variations archive (NCBI), rs855791 identifies T>C substitution in position 2207 (correspondent to a complementary A>G change), resulting the missense valine (V) to alanine (A) change in position 736 (p.V736A variant), nearby catalytic and binding sites of MT-2. The human database indicates V at the 736 position as the “wild type” amino acid of MT-2; A is evolutionary conserved in other species, suggesting it is the ancestral amino acid and that V in position 736 is a recent evolutionary change [31]. In this study we investigated the presence and the role of the TMPRSS6 polymorphisms in patients with IDA, referred to our outpatients service because only partially responsive to oral iron therapy. Fifty healthy blood donors were used as control group. TMPRSS6 sequencing has been performed with these aims: a) to identify TMPRSS6 polymorphisms in enrolled population and in healthy control group; b) to identify cases of pure IRIDA.
METHODS Study subjects Between January 2009 and December 2013, 113 subjects (12 males, 101 females) with mean age 36 ± 13 years (range 4-74 years) were referred to the Rare Diseases Centre of “Fondazione IRCCS Ca’ Granda, Ospedale Maggiore Policlinico di Milano” for persistent IDA poorly responsive to oral iron. All the subjects were previously treated with oral iron treatment by their general practitioners or other hematologists with incomplete response even in presence of good patient’s compliance and proper treatment dose and duration. Most of the patients reported a mild degree of iron deficiency anemia during childhood or adolescence, treated with repeated cycles of oral iron therapy, but 3 This article is protected by copyright. All rights reserved.
always with partial benefits. All the patients reported normal growth and a good quality of life. In all the cases we investigated celiac disease, gastrointestinal bleeding and Helicobacter Pylori (HP) infection (Figure 1). Hematologic parameters, iron status, inflammatory markers and thyroid function were tested. Fifty healthy donors ethnic-matched to the subjects enrolled in the study, were used as control groups (Table I). Sequence variation in TMPRSS6 gene was evaluated by PCR and direct sequencing in genomic DNA isolated from peripheral lymphomonocytes. Direct sequencing was performed using a fluorescence-tagged dideoxy chain terminator method in an ABI BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA, USA), according to the manufacturer’s instructions. Thalassemia trait was suspected and investigated in 38 out of 113 patients (34%; 3 males, 35 females) using HPLC and molecular analysis of globin genes. Beta globin chain gene has been investigated for the common Mediterranean mutations, including cod. 39, cod. 6, IVS1-1, IVS2-1 (β°), and -101, -87, IVS1-6, IVS1-110, IVS2-745 (β+), by DGGE (Denaturing Gradient Gel Electroforesis) after PCR (polymerase chain reaction) DNA amplification. The analysis of α-globin genes has been performed in selected patients. The study has been conducted according to Helsinki Declaration. All the recruited patients have given their signed informed consent. For statistical analysis, multiple linear regression models adjusted for sex and age (continuous) was used (Armitage P, Berry G, Matthews JNS. Statistical methods in medical research; Fourth Edition, Oxford, Blackwell Science 2002) as well as multiple logistic regression models were used (Clayton D, Hills M. Statistical models in epidemiology, Oxford; New York: Oxford University Press, 1993). We calculated sex- and age-adjusted odds ratios (OR) and 95% confidence intervals (95% CI) of being a case for V736A homozygosis, heterozygosis and wild type. When OR was not estimable because of zero controls, we reported the p-value from chi-square test. Analyses were performed with Stata 13 (StataCorp. 2013. Stata: Release 13. Statistical Software. College Station, TX: StataCorp LP).
RESULTS The hematologic characteristics and iron status of patients and controls are reported in Table I. Considering the common TMPRSS6 polymorphisms, we found that the frequencies of V736A, SNP-120, SNP-113, F5F, P33P, K253E, S361S, Y418Y, D521D, Δ15accc and Y739Y were markedly different between patients and controls (Figure 2, Table II). Homozigosity for V736A was detected in 40 patients (35.5%), whereas it was never detected in healthy controls; forty-eight (42.5%) patients resulted heterozygotes, as reported in Figure 2.
4 This article is protected by copyright. All rights reserved.
Hematologic parameters in enrolled population according to genotype (homozygotes, heterozygotes and wild type for V736A) are reported in Table III. Considering together patients and controls, we found that the presence of the polymorphism V736A both in heterozygosis and in homozygosis is associated to lower hemoglobin levels (respectively, -0.6 gr/dL, p=0.05; and -0.9 gr/dL, p=0.01) as well as a statistically significant variation in MCV and MCH values was observed (Table IV). Agreement percentage between V736A and other polymorphisms in whole population are reported in Table V. The relationship with the common polymorphisms D521D and Y739Y are shown in Tables VI and VII. A new variant - H448R - was identified in a patient with IDA requiring parenteral iron supplementation. Two uncommon polymorphisms, A719T and V795I (estimated frequency
