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Idiopathic pulmonary fibrosis and polymorphisms of the folate pathway genes
Clinical Biochemistry 46 (2013) 85–88
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Idiopathic pulmonary fibrosis and polymorphisms of the folate pathway genes Marcella Martinelli a, b, 1, Luca Scapoli a, b, 1, Paolo Carbonara c, Ilaria Valentini c, Ambra Girardi a, Francesca Farinella d, Gabriella Mattei a, b, Angela Maria Grazia Pacilli c, Luca Fasano e, Stefano Nava e, Rossella Solmi a, b,⁎ a
Dipartimento di Istologia, Embriologia e Biologia Applicata, Università di Bologna, Via Belmeloro 8, 40126 Bologna, Italy Centro di Ricerca in Genetica Molecolare “Fondazione CARISBO,” Bologna, Italy Dipartimento di Medicina Interna dell'Invecchiamento e Malattie Nefrologiche, Università di Bologna, via Massarenti, 9, 40138 Bologna, Italy d Dipartimento di Discipline Medico Chirurgiche della Comunicazione e del Comportamento, Corso Giovecca 203, 44121 Ferrara, Italy e Dipartimento Cardio-Toraco-Vascolare, UO Pneumologia e TI Respiratoria, Policlinico S.Orsola-Malpighi, via Massarenti, 9, 40138 Bologna, Italy b c
a r t i c l e
i n f o
Article history: Received 14 May 2012 received in revised form 26 July 2012 accepted 9 October 2012 Available online 23 October 2012 Keywords: Idiopathic pulmonary fibrosis Polymorphisms Folate pathway
a b s t r a c t Objectives: This study aims to determine the possible association between folate pathway gene polymorphisms and idiopathic pulmonary fibrosis. This represents the first study carried out on folate pathway gene polymorphisms as possible risk factors in this kind of pathology. The premise is that several polymorphisms mapping on genes responsible for folate uptake are associated with the risk of numerous diseases occurring between pregnancy and old age, and that too little is currently known about idiopathic pulmonary fibrosis. Design and methods: We genotyped 9 single nucleotide polymorphisms and 1 polymorphic insertion in 7 essential genes belonging to the folate pathway in 32 Italian idiopathic pulmonary fibrosis patients and 81 control subjects. This was done by PCR and restriction analysis. Results: Allelic and genotypic association tests indicated that for all the analysed polymorphisms there were no significant differences between patients and controls. Nevertheless, the haplotype association analysis revealed a significant association between idiopathic pulmonary fibrosis and transcobalamin II gene polymorphisms: specifically the haplotype 776G (rs1801198)–c.1026-394G (rs7286680)–444C (rs10418) (OR=2.84; 95% C.I. 1.36–5.93, P value=0.004). Conclusions: This small-scale preliminary study would suggest the importance of further research focusing on the role of folate in the onset of idiopathic pulmonary fibrosis. © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Introduction “Idiopathic pulmonary fibrosis (IPF) is the most common and most lethal diffuse fibrosing lung disease, with a mortality rate that exceeds that of many cancers” [1]. No studies have previously considered a possible relationship between defective folate uptake and IPF even if, in this pathology, the altered DNA synthesis is a crucial crossroads. In fact, IPF is characterized by initial alveolar epithelial injury, infiltration of inflammatory cells, abnormal tissue repair [2] with fibroblast hyperplasia, deposition of extracellular matrix and scar formation [3,4]. The architecture of the lung is disrupted, giving it a “honeycomb” appearance [3]. The repeated micro injuries which occur over time to different areas of the lung are referred to as usual interstitial pneumonia (UIP) [1].
⁎ Corresponding author at: Dipartimento di Istologia, Embriologia e Biologia Applicata, Via Belmeloro, 8 40126 Bologna, Italy. Fax: +39 051 2094110. E-mail address: [email protected] (R. Solmi). 1 These authors are equal contributors.
Folates are essential water-soluble B vitamins that function as coenzymes, primarily in reactions for the biosynthesis of nucleotides and methionine. Several polymorphisms (SNPs) of the folate uptake genes are associated with the risk of a certain number of diseases occurring from pregnancy (congenital malformations: cleft lip [5], neural tube defects [6]), to middle and old age (lung cancer [7]). Direct and indirect evidence seems to correlate folate uptake with IPF: 1) Alveolar and interstitial inflammatory macrophages that play a key role in the pathogenesis of idiopathic pulmonary fibrosis (producing proinflammatory and/or fibrogenic cytokines) express folate receptor β differently to resident macrophages in normal tissues [4]. Folate receptors are expressed in addition to reduced folate carriers and proton-coupled folate transporters, and mediate the unidirectional transport of folates into cells. Four isoforms of folate receptors were identified (α, β, γ, δ or 1, 2, 3, 4, respectively). β (as well as α) isoform is a glycosylphosphatidylinositol-anchored glycoprotein with a high affinity for folic acid and 5-methyltetrahydrofolate [8]. It is expressed in the placenta and in some hematopoietic cells of the myelogenous lineage [9].
0009-9120/$ – see front matter © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.clinbiochem.2012.10.009
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M. Martinelli et al. / Clinical Biochemistry 46 (2013) 85–88
2) Some of the risk factors associated with IPF, like cigarette smoking and the effects of ageing – which lead to the abnormal reconstitution of the epithelial barrier [1] – are strictly related to difficulties in absorbing folic acid from food. 3) At present, there are no established therapies for IPF [10]. Current therapies (azathiprine, corticosteroids, cyclophosphamide, pirfenidone) based on anti-inflammatory, anti-fibrosis or immunosuppressive treatments are useless. Antioxidant strategies and other therapeutic agents targeting fibrogenesis – such as interferons, antagonists of cytokines, growth factors or their receptors, and angiogenic agents – have been tried out, but have produced no satisfactory results [11]. Cortisone-based drugs, in particular, block folic acid absorption. Finally, recent studies confirm a genetic basis in the development of IPF. For example, heterozygous mutations were found in: the genes that encode the protein component of telomerase (TERT) [12–14], the surfactant proteins C and A2 [12,15], and the promoter of MUC5B gene, that encodes a highly glycosylated macromolecular component of mucus secretions [16]. Several mediators – including the fibroblast transforming growth factor β1 (TGF-β1), cytokines, metalloproteinases [1], altered surfactant proteins [12] and secreted airway mucins [16] – influence the histopathological process that defines idiopathic pulmonary fibrosis; but the recently found mutations are not sufficient to explain the complex aetiology of this serious disease. This study is the first to investigate several important polymorphisms of genes belonging to the folate pathway as possible risk factors for IPF. Folate could be an important element in pulmonary homeostasis and its correct assumption therefore essential to ensure DNA stability. In order to find a potential effective therapy we need to better understand the role of folate uptake in IPF. Subjects and methods Subjects Between 2008 and 2011, 32 consecutive Italian patients with IPF (19 males and 13 females) were enlisted for this study at the Respiratory Division, Sant'Orsola Hospital, University of Bologna. Seventeen of these patients were ex-smokers. Diagnosis of IPF was made according to new ATS/ERS guidelines [17]: in particular, identifiable causes of interstitial lung disease were excluded, and all patients underwent high resolution thoracic CT demonstrating a UIP pattern or possible UIP pattern. Lung biopsy, in order to confirm diagnosis, was required in only 10 subjects. The patients underwent lung function testing including determination of resting arterial blood gases in room air (using a Radiometer ABL 520 Blood Gas Analyzer, Radiometer Medical ApS, Copenhagen, Denmark), spirometry and DLCO (using a Morgan Pulmolab 544, Ferraris Respiratory Europe Ltd, Hertford, UK). Arterial blood sampling in room air was not available for three patients, whose clinical conditions did not allow for their oxygen therapy to be suspended. A high resolution CT scan was also performed, using a Light Speed QX General Electric CT (General Electric, Milwaukee, Wisconsin, USA), to determine automatic fibrosis scores (volume of fibrosis/total lung volume %). Eighty-one unrelated volunteers, with a healthy general clinical and biochemical assessment, were used as a control group for this investigation. All patient and control group information is summarised in Table 1. The study was approved by the ethical committee of Sant'OrsolaMalpighi General Hospital and complied with the Ethical Principles for Medical Research Involving Human Subjects of the Helsinki Declaration. Written informed consent was obtained from all patients and healthy controls beforehand. Peripheral blood samples were drawn from each individual and DNA extraction was performed using the GenElute Blood Genomic DNA kit (SIGMA, Milan, Italy).
Table 1 Patient and control subjects. Characteristics
Patientse
Controlse
P value
Age years Age at diagnosis Gender Female Male Functional and radiological features PaO2 (mmHg)a DLCO (% of pred)b FVC (% of pred)c CT automatic score (% of total lung volume)d Pattern CT
68.2 ± 9.7 65.2 ± 9.4
68 ± 12.6 –
0.93
13 17
38 43
69.7 ± 16.0 42.2 ± 21.4 81.7 ± 28.1 32.9 ± 14.8
– – – –
UIP/NSIP 21 11
–
a b c d e
0.19
PaO2 partial pressure of oxygen in arterial blood. DLCO carbon monoxide diffusing capacity of the lung. FVC forced vital capacity. CT computed tomography. All values are expressed as average and standard deviation.
Genetic analysis We tested the following polymorphisms: A401G (rs1950902) and G1958A (rs2236225) for methylenetetrahydrofolate dehydrogenase 1 (MTHFD1); A2756G (rs1805087) for 5-methyltetrahydrofolatehomocysteine methyltransferase (MTR); C776G (rs1801198), C444T (rs10418), and c.1026-394T>G (rs7286680) for transcobalamin II (TCN2); G80A (rs1051266) for solute carrier family 19 (folate transporter), member 1 (RFC1/SLC19A1); C665T (rs1801133) for methylenetetrahydrofolate reductase (MTHFR); c.243-1008A > G (rs1643659) for dihydrofolate reductase (DHFR), and the polymorphism 844ins68 mapping in cystathionine beta synthase (CBS). The polymorphism choice was based on literature data and included those with a known or suspected functional impact, such as SNPs associated with other diseases and missense polymorphisms. We performed SNP genotyping by PCR followed by restriction analysis and separation of fragments by 10% polyacrylamide gel electrophoresis stained with ethidium bromide. The restriction enzymes employed were MspI, BsmAI, HaeIII, BstNI, AvaII, HaeIII, HaeII and BstNI (New England Biolabs, Milan, Italy) respectively. The CBS 68 bp insertion presence was assessed by electrophoresis run on 2.5% agarose of amplicons. Statistical analysis The distribution of genotypes in the patient and control groups was tested for deviations from the Hardy–Weinberg equilibrium using Pearson's χ2 test. Genetic association was investigated using both allelic and genotypic tests with a likelihood ratio approach by unphased software v3.1.5 using a Windows Vista operative system [18]. Odds ratios were calculated in order to evaluate the level of association of the rare allele carriers, as well as of heterozygotes and homozygotes. Haplotype association was evaluated for the TCN2 gene because three gene polymorphisms were available. A global association test that reveals all associated haplotypes was performed, as well as a specific test for each haplotype. A permutation test was conducted with 1,000 sample replicates, where the trait values were randomly shuffled between subjects to allow for multiple testing corrections over all tests performed. Results A high success rate was obtained during the genotyping step for all the different assays. Genotype frequencies among both cases and controls were in agreement with the Hardy–Weinberg law.
M. Martinelli et al. / Clinical Biochemistry 46 (2013) 85–88
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Table 2 SNP characteristics, allele count and allelic association test. Gene and polymorphism ID
CBS 844ins68 MTHFD1 rs2236225 rs1950902 MTR rs1805087 TCN2 rs1801198 rs7286680 rs10418 RFC1 rs1051266 MTHFR rs1801133 DHFR rs1643659 a b
Polymorphism characteristics
Case
Nucleotide changea
Location or amino acid substitution
–
Exon 8
G/A G/A
Arg653Gln Arg134Lys
A/G
Assay ID/restriction site
Control
Allelic association
A (%)
a (%)
A (%)
a (%)
P value
OR (95% C.I.)
61 (95)
3 (05)
152 (95)
8 (05)
0.92
0.93 (0.24–3.64)
MspI BsmAI
32 (52) 55 (86)
30 (48) 9 (14)
81 (51) 141 (88)
79 (49) 19 (12)
0.89 0.66
0.96 (0.53–1.73) 1.21 (0.52–2.85)
Asp919Gly
HaeIII
55 (86)
9 (14)
124 (78)
36 (23)
0.14
0.56 (0.25–1.25)
C/G T/G C/T
Pro259Arg Intron 3′-UTR
BstNI HaeIII AvaIIb
26 (41) 35 (55) 54 (87)
38 (59) 29 (45) 8 (13)
90 (56) 106 (65) 133 (83)
72 (44) 56 (35) 27 (17)
0.04 0.14 0.46
1.83 (1.02–3.29) 1.57 (0.87–2.83) 0.73 (0.31–1.71)
A/G
His27Arg
HaeII
37 (58)
27 (42)
84 (53)
76 (48)
0.47
0.81 (0.45–1.45)
C/T
Ala222Val
HinfI
37 (58)
27 (42)
84 (52)
78 (48)
0.42
0.79 (0.44–1.41)
A/G
Intron
BstNI
38 (59)
26 (41)
109 (68)
51 (32)
0.22
1.46 (0.80–2.66)
Major allele/minor. An artificial restriction site for AvaII was created by using a mutagenic sense primer with a mismatch in the nucleotide sequence.
The allelic association test revealed that allele frequencies were similar in case and control groups (Table 2), while for the rs1801198, in TCN2, marginal values of association were detected. Indeed, the variant allele 776G was more frequent among patients with IPF: the calculated odds ratio was 1.83 (95% C.I. 1.02–3.29; P value= 0.04). However, the permutation analysis by multiple testing correction indicated that this difference was not significant (P value = 0.39). Genotypic association testing provided similar results (Table 3). The rs1801198 polymorphism showed an overall genotypic association close to the nominal levels of significance (P value = 0.06), and an increased risk for the homozygote carrier of the rare allele, OR= 5.25 (95% C.I. 1.19–23.2). The haplotype association analysis, that was conducted for the three polymorphisms of TCN2, supported a genetic association between IPF and TCN2. Indeed, the overall statistics for testing whether any haplotypes were associated, provided evidence of association with threemarker haplotype analysis (P value= 0.04). The specific association values and estimated odds ratio for each of the haplotypes are reported in Table 4. The haplotype 776G (rs1801198)–c.1026-394G (rs7286680)–444C (rs10418) showed a significant association with IPF OR = 2.84 (95% C.I. 1.36–5.93, P value = 0.004), which remained significant after multiple testing correction (P value = 0.04). Discussion IPF is a multistep disease influenced by a variety of cellular alterations. During the first step of IPF, in a restricted pulmonary area,
unknown endogenous and/or environmental stimuli disrupt alveolar epithelial cells, causing abnormal epithelial repair [19]. The alveolar epithelium is normally composed of two types of pneumocytes: type I and type II. Type I are squamous in form and cover the majority of the alveolar surface area (>95%). They are responsible for gas exchange in the alveoli, are unable to replicate, and are susceptible to toxic insults. Type II pneumocytes are the cuboidal and granular cells at the alveolar-septal junction. They cover about 5% of the total alveolar surface area and, in the event of damage, can proliferate and/or differentiate into type I cells. Type II cells are responsible for the production and secretion of surfactant (dipalmitoylphosphatidylcholine), a group of phospholipids that reduce the alveolar surface tension. Several recently-published studies regarding the risk of an association of IPF with SNPs of genes coding MUC5B [16] and the surfactant C and A2 proteins [12,15] revealed genetic bases of IPF linked to a consequent functional alteration of the type II cells which produce these proteins. The main focus of this introductory study (which aims to analyze the SNPs of the folate pathway genes in patients with IPF) is on activated macrophages (another cell type with a key role in IPF) because, in the injured area of alveolar tissue, they express folate receptor β, showing the need for major folate uptake [20]. Cells in rapid division require abundant amounts of folate, essential for the biosynthesis of nucleotides and for methylation [20]. Macrophages continuously clean dirt and microrganisms from the alveolar surface. Cytokines or fragments of pathogenic microbes stimulate the activation of macrophages, giving them enhanced ability to intervene against microrganisms [21]. Activated macrophages also
Table 3 Genotype distribution and overall genotypic association. Gene
Polymorphism ID
CBS MTHFD1 MTHFD1 MTR TCN2 TCN2 TCN2 RFC1 MTHFR DHFR
844ins68 rs2236225 rs1950902 rs1805087 rs1801198 rs7286680 rs10418 rs1051266 rs1801133 rs1643659
Case
Control
Overall assoc.
Heterozygote
Homozygote
AA
Aa
aa
AA
Aa
aa
P value
OR (95% C.I.)
OR (95% C.I.)
29 8 24 25 3 9 23 11 11 11
3 16 7 5 20 17 8 15 15 16
0 7 1 2 9 6 0 6 6 5
73 21 61 50 21 35 56 19 21 35
6 39 19 24 48 36 21 46 42 39
1 20 0 6 12 10 3 15 18 6
0.68 0.95 0.28 0.24 0.06 0.30 0.36 0.49 0.67 0.38
1.26 1.08 0.94 0.42 2.92 1.84 0.93 0.56 0.68 1.31
n.d. 0.92 n.d. 0.67 5.25 2.33 n.d. 0.69 0.64 2.65
(0.29–5.37) (0.40–2.93) (0.35–2.51) (0.14–1.22) (0.78–10.9) (0.72–4.67) (0.36–2.39) (0.22–1.45) (0.27–1.74) (0.53–3.19)
(0.28–3.01) (0.13–3.54) (1.19–23.2) (0.67–8.14) (0.21–2.30) (0.20–2.07) (0.68–10.4)
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M. Martinelli et al. / Clinical Biochemistry 46 (2013) 85–88
Table 4 Haplotype association analysis between TCN2 polymorphisms and IPF. Haplotypes are combination of rs1801198, rs7286680, and rs10418 alleles. Haplotype
C–T-C G–T–C G–G–C G–G–T
Case
Control
Association
n
(%)
n
(%)
P value
OR (95% C.I.)
25 8 21 8
(40) (13) (34) (13)
81 21 24 22
(55) (14) (16) (15)
0.06 0.81 0.004 0.71
1 1.23 (0.49–3.13) 2.84 (1.36–5.93) 1.18 (0.47–2.97)
acquire opposing roles [20,22] depending on the stimulus: in autoimmune diseases (rheumatoid arthritis, lupus, atherosclerosis, psoriasis, diabetes, and transplant rejection) they can cause severe tissue damage. Proinflammatory or profibrotic cytokines produced by them – specifically the macrophage colony-stimulating factor (important for cell proliferation and survival) and TGF-β1 (directly linked to fibrosis) [23] – have higher values in patients with IPF than in patients with other kinds of pulmonary diseases [4,24]. It is believed that macrophages can act on local lung fibroblasts and possibly also recruit fibrocytes [4,25]. An altered proliferation of fibroblasts substituting destroyed pneumocytes causes loss of alveolar function. The same process, occurring in other areas at different times, eventually damages the lungs. The origin of such fibroblasts has not been identified; however, they may be recruited after an epithelial–mesenchymal transition (EMT) [26,27]. It is essential to clarify the role of folate in IPF, given the aberrant proliferation of fibroblasts and particularly of activated macrophages. Although it might seem necessary to prevent folate absorption in order to reduce the proliferation of these cells, it has been proven that cortisone-based drugs, which obstruct folate absorption, do not achieve positive effects. Moreover, cigarette smoking, which also obstructs folate absorption, is itself a risk factor. On the other hand, a therapeutic strategy worth considering could be folate combined with medications. In fact, β receptors of activated macrophages could link with the folate, thereby allowing the other substances in the medication to act at the source of the fibrosis. Our study has a number of limitations. A multicenter study and/or a larger study of an independent patient population would be needed to have an adequate sample size to which to refer. Evaluation of the folate uptake and the folate/B12/homocysteine status of patients with IPF would also be necessary in order to correlate these values with the presence of SNPs in the folate pathway genes. The data obtained from the present investigation indicate no significant differences between patients and control subjects regarding the variant alleles of the SNPs analysed, but the haplotype 776G (rs1801198)–c.1026-394G (rs7286680)–444C (rs10418) for the TCN2 gene showed a significant association with IPF. Our hypothesis that SNPs located on key genes of the folate pathway could exert a possible influence on folate absorption was not confirmed by our results. However, a larger more comprehensive study is required to fully address the question of the real role of SNPs of the folate pathway in IPF. Finally, this introductory study is of significance in order to recognize the importance of further research into the potential therapeutic value of folate–drug conjugates (like those studied for cancer and inflammatory pathologies [22,28,29]) to combat the aberrant proliferation of activated macrophages.
Acknowledgments We are grateful to Dr. Maria Carmen Biffoni and Dr. Daniela Solmi (Laboratorio Analisi Cliniche S. Antonio, Bologna) for their precious collaboration in supplying blood samples from healthy donors. We are indebted to all the patients who agreed to participate in the study.
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Single nucleotide polymorphisms in the transcobalamin gene: relationship with transcobalamin concentrations and risk for neural tube defects. Eur J Hum Genet 2002;10:433-8. [7] Stidley CA, Picchi MA, Leng S, Willink R, Crowell RE, Flores KG, et al. Multivitamins, folate, and green vegetables protect against gene promoter methylation in the aerodigestive tract of smokers. Cancer Res 2010;70:568-74. [8] Antony AC. Folate receptors. Annu Rev Nutr 1996;16:501-21. [9] Reddy JA, Haneline LS, Srour EF, Antony AC, Clapp DW, Low PS. Expression and functional characterization of the beta-isoform of the folate receptor on cd34(+) cells. Blood 1999;93:3940-8. [10] Sisson TH, Mendez M, Choi K, Subbotina N, Courey A, Cunningham A, et al. Targeted injury of type ii alveolar epithelial cells induces pulmonary fibrosis. Am J Respir Crit Care Med 2010;181:254-63. [11] Farkas L, Gauldie J, Voelkel NF, Kolb M. 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Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem
Idiopathic pulmonary fibrosis and polymorphisms of the folate pathway genes Marcella Martinelli a, b, 1, Luca Scapoli a, b, 1, Paolo Carbonara c, Ilaria Valentini c, Ambra Girardi a, Francesca Farinella d, Gabriella Mattei a, b, Angela Maria Grazia Pacilli c, Luca Fasano e, Stefano Nava e, Rossella Solmi a, b,⁎ a
Dipartimento di Istologia, Embriologia e Biologia Applicata, Università di Bologna, Via Belmeloro 8, 40126 Bologna, Italy Centro di Ricerca in Genetica Molecolare “Fondazione CARISBO,” Bologna, Italy Dipartimento di Medicina Interna dell'Invecchiamento e Malattie Nefrologiche, Università di Bologna, via Massarenti, 9, 40138 Bologna, Italy d Dipartimento di Discipline Medico Chirurgiche della Comunicazione e del Comportamento, Corso Giovecca 203, 44121 Ferrara, Italy e Dipartimento Cardio-Toraco-Vascolare, UO Pneumologia e TI Respiratoria, Policlinico S.Orsola-Malpighi, via Massarenti, 9, 40138 Bologna, Italy b c
a r t i c l e
i n f o
Article history: Received 14 May 2012 received in revised form 26 July 2012 accepted 9 October 2012 Available online 23 October 2012 Keywords: Idiopathic pulmonary fibrosis Polymorphisms Folate pathway
a b s t r a c t Objectives: This study aims to determine the possible association between folate pathway gene polymorphisms and idiopathic pulmonary fibrosis. This represents the first study carried out on folate pathway gene polymorphisms as possible risk factors in this kind of pathology. The premise is that several polymorphisms mapping on genes responsible for folate uptake are associated with the risk of numerous diseases occurring between pregnancy and old age, and that too little is currently known about idiopathic pulmonary fibrosis. Design and methods: We genotyped 9 single nucleotide polymorphisms and 1 polymorphic insertion in 7 essential genes belonging to the folate pathway in 32 Italian idiopathic pulmonary fibrosis patients and 81 control subjects. This was done by PCR and restriction analysis. Results: Allelic and genotypic association tests indicated that for all the analysed polymorphisms there were no significant differences between patients and controls. Nevertheless, the haplotype association analysis revealed a significant association between idiopathic pulmonary fibrosis and transcobalamin II gene polymorphisms: specifically the haplotype 776G (rs1801198)–c.1026-394G (rs7286680)–444C (rs10418) (OR=2.84; 95% C.I. 1.36–5.93, P value=0.004). Conclusions: This small-scale preliminary study would suggest the importance of further research focusing on the role of folate in the onset of idiopathic pulmonary fibrosis. © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Introduction “Idiopathic pulmonary fibrosis (IPF) is the most common and most lethal diffuse fibrosing lung disease, with a mortality rate that exceeds that of many cancers” [1]. No studies have previously considered a possible relationship between defective folate uptake and IPF even if, in this pathology, the altered DNA synthesis is a crucial crossroads. In fact, IPF is characterized by initial alveolar epithelial injury, infiltration of inflammatory cells, abnormal tissue repair [2] with fibroblast hyperplasia, deposition of extracellular matrix and scar formation [3,4]. The architecture of the lung is disrupted, giving it a “honeycomb” appearance [3]. The repeated micro injuries which occur over time to different areas of the lung are referred to as usual interstitial pneumonia (UIP) [1].
⁎ Corresponding author at: Dipartimento di Istologia, Embriologia e Biologia Applicata, Via Belmeloro, 8 40126 Bologna, Italy. Fax: +39 051 2094110. E-mail address: [email protected] (R. Solmi). 1 These authors are equal contributors.
Folates are essential water-soluble B vitamins that function as coenzymes, primarily in reactions for the biosynthesis of nucleotides and methionine. Several polymorphisms (SNPs) of the folate uptake genes are associated with the risk of a certain number of diseases occurring from pregnancy (congenital malformations: cleft lip [5], neural tube defects [6]), to middle and old age (lung cancer [7]). Direct and indirect evidence seems to correlate folate uptake with IPF: 1) Alveolar and interstitial inflammatory macrophages that play a key role in the pathogenesis of idiopathic pulmonary fibrosis (producing proinflammatory and/or fibrogenic cytokines) express folate receptor β differently to resident macrophages in normal tissues [4]. Folate receptors are expressed in addition to reduced folate carriers and proton-coupled folate transporters, and mediate the unidirectional transport of folates into cells. Four isoforms of folate receptors were identified (α, β, γ, δ or 1, 2, 3, 4, respectively). β (as well as α) isoform is a glycosylphosphatidylinositol-anchored glycoprotein with a high affinity for folic acid and 5-methyltetrahydrofolate [8]. It is expressed in the placenta and in some hematopoietic cells of the myelogenous lineage [9].
0009-9120/$ – see front matter © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.clinbiochem.2012.10.009
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2) Some of the risk factors associated with IPF, like cigarette smoking and the effects of ageing – which lead to the abnormal reconstitution of the epithelial barrier [1] – are strictly related to difficulties in absorbing folic acid from food. 3) At present, there are no established therapies for IPF [10]. Current therapies (azathiprine, corticosteroids, cyclophosphamide, pirfenidone) based on anti-inflammatory, anti-fibrosis or immunosuppressive treatments are useless. Antioxidant strategies and other therapeutic agents targeting fibrogenesis – such as interferons, antagonists of cytokines, growth factors or their receptors, and angiogenic agents – have been tried out, but have produced no satisfactory results [11]. Cortisone-based drugs, in particular, block folic acid absorption. Finally, recent studies confirm a genetic basis in the development of IPF. For example, heterozygous mutations were found in: the genes that encode the protein component of telomerase (TERT) [12–14], the surfactant proteins C and A2 [12,15], and the promoter of MUC5B gene, that encodes a highly glycosylated macromolecular component of mucus secretions [16]. Several mediators – including the fibroblast transforming growth factor β1 (TGF-β1), cytokines, metalloproteinases [1], altered surfactant proteins [12] and secreted airway mucins [16] – influence the histopathological process that defines idiopathic pulmonary fibrosis; but the recently found mutations are not sufficient to explain the complex aetiology of this serious disease. This study is the first to investigate several important polymorphisms of genes belonging to the folate pathway as possible risk factors for IPF. Folate could be an important element in pulmonary homeostasis and its correct assumption therefore essential to ensure DNA stability. In order to find a potential effective therapy we need to better understand the role of folate uptake in IPF. Subjects and methods Subjects Between 2008 and 2011, 32 consecutive Italian patients with IPF (19 males and 13 females) were enlisted for this study at the Respiratory Division, Sant'Orsola Hospital, University of Bologna. Seventeen of these patients were ex-smokers. Diagnosis of IPF was made according to new ATS/ERS guidelines [17]: in particular, identifiable causes of interstitial lung disease were excluded, and all patients underwent high resolution thoracic CT demonstrating a UIP pattern or possible UIP pattern. Lung biopsy, in order to confirm diagnosis, was required in only 10 subjects. The patients underwent lung function testing including determination of resting arterial blood gases in room air (using a Radiometer ABL 520 Blood Gas Analyzer, Radiometer Medical ApS, Copenhagen, Denmark), spirometry and DLCO (using a Morgan Pulmolab 544, Ferraris Respiratory Europe Ltd, Hertford, UK). Arterial blood sampling in room air was not available for three patients, whose clinical conditions did not allow for their oxygen therapy to be suspended. A high resolution CT scan was also performed, using a Light Speed QX General Electric CT (General Electric, Milwaukee, Wisconsin, USA), to determine automatic fibrosis scores (volume of fibrosis/total lung volume %). Eighty-one unrelated volunteers, with a healthy general clinical and biochemical assessment, were used as a control group for this investigation. All patient and control group information is summarised in Table 1. The study was approved by the ethical committee of Sant'OrsolaMalpighi General Hospital and complied with the Ethical Principles for Medical Research Involving Human Subjects of the Helsinki Declaration. Written informed consent was obtained from all patients and healthy controls beforehand. Peripheral blood samples were drawn from each individual and DNA extraction was performed using the GenElute Blood Genomic DNA kit (SIGMA, Milan, Italy).
Table 1 Patient and control subjects. Characteristics
Patientse
Controlse
P value
Age years Age at diagnosis Gender Female Male Functional and radiological features PaO2 (mmHg)a DLCO (% of pred)b FVC (% of pred)c CT automatic score (% of total lung volume)d Pattern CT
68.2 ± 9.7 65.2 ± 9.4
68 ± 12.6 –
0.93
13 17
38 43
69.7 ± 16.0 42.2 ± 21.4 81.7 ± 28.1 32.9 ± 14.8
– – – –
UIP/NSIP 21 11
–
a b c d e
0.19
PaO2 partial pressure of oxygen in arterial blood. DLCO carbon monoxide diffusing capacity of the lung. FVC forced vital capacity. CT computed tomography. All values are expressed as average and standard deviation.
Genetic analysis We tested the following polymorphisms: A401G (rs1950902) and G1958A (rs2236225) for methylenetetrahydrofolate dehydrogenase 1 (MTHFD1); A2756G (rs1805087) for 5-methyltetrahydrofolatehomocysteine methyltransferase (MTR); C776G (rs1801198), C444T (rs10418), and c.1026-394T>G (rs7286680) for transcobalamin II (TCN2); G80A (rs1051266) for solute carrier family 19 (folate transporter), member 1 (RFC1/SLC19A1); C665T (rs1801133) for methylenetetrahydrofolate reductase (MTHFR); c.243-1008A > G (rs1643659) for dihydrofolate reductase (DHFR), and the polymorphism 844ins68 mapping in cystathionine beta synthase (CBS). The polymorphism choice was based on literature data and included those with a known or suspected functional impact, such as SNPs associated with other diseases and missense polymorphisms. We performed SNP genotyping by PCR followed by restriction analysis and separation of fragments by 10% polyacrylamide gel electrophoresis stained with ethidium bromide. The restriction enzymes employed were MspI, BsmAI, HaeIII, BstNI, AvaII, HaeIII, HaeII and BstNI (New England Biolabs, Milan, Italy) respectively. The CBS 68 bp insertion presence was assessed by electrophoresis run on 2.5% agarose of amplicons. Statistical analysis The distribution of genotypes in the patient and control groups was tested for deviations from the Hardy–Weinberg equilibrium using Pearson's χ2 test. Genetic association was investigated using both allelic and genotypic tests with a likelihood ratio approach by unphased software v3.1.5 using a Windows Vista operative system [18]. Odds ratios were calculated in order to evaluate the level of association of the rare allele carriers, as well as of heterozygotes and homozygotes. Haplotype association was evaluated for the TCN2 gene because three gene polymorphisms were available. A global association test that reveals all associated haplotypes was performed, as well as a specific test for each haplotype. A permutation test was conducted with 1,000 sample replicates, where the trait values were randomly shuffled between subjects to allow for multiple testing corrections over all tests performed. Results A high success rate was obtained during the genotyping step for all the different assays. Genotype frequencies among both cases and controls were in agreement with the Hardy–Weinberg law.
M. Martinelli et al. / Clinical Biochemistry 46 (2013) 85–88
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Table 2 SNP characteristics, allele count and allelic association test. Gene and polymorphism ID
CBS 844ins68 MTHFD1 rs2236225 rs1950902 MTR rs1805087 TCN2 rs1801198 rs7286680 rs10418 RFC1 rs1051266 MTHFR rs1801133 DHFR rs1643659 a b
Polymorphism characteristics
Case
Nucleotide changea
Location or amino acid substitution
–
Exon 8
G/A G/A
Arg653Gln Arg134Lys
A/G
Assay ID/restriction site
Control
Allelic association
A (%)
a (%)
A (%)
a (%)
P value
OR (95% C.I.)
61 (95)
3 (05)
152 (95)
8 (05)
0.92
0.93 (0.24–3.64)
MspI BsmAI
32 (52) 55 (86)
30 (48) 9 (14)
81 (51) 141 (88)
79 (49) 19 (12)
0.89 0.66
0.96 (0.53–1.73) 1.21 (0.52–2.85)
Asp919Gly
HaeIII
55 (86)
9 (14)
124 (78)
36 (23)
0.14
0.56 (0.25–1.25)
C/G T/G C/T
Pro259Arg Intron 3′-UTR
BstNI HaeIII AvaIIb
26 (41) 35 (55) 54 (87)
38 (59) 29 (45) 8 (13)
90 (56) 106 (65) 133 (83)
72 (44) 56 (35) 27 (17)
0.04 0.14 0.46
1.83 (1.02–3.29) 1.57 (0.87–2.83) 0.73 (0.31–1.71)
A/G
His27Arg
HaeII
37 (58)
27 (42)
84 (53)
76 (48)
0.47
0.81 (0.45–1.45)
C/T
Ala222Val
HinfI
37 (58)
27 (42)
84 (52)
78 (48)
0.42
0.79 (0.44–1.41)
A/G
Intron
BstNI
38 (59)
26 (41)
109 (68)
51 (32)
0.22
1.46 (0.80–2.66)
Major allele/minor. An artificial restriction site for AvaII was created by using a mutagenic sense primer with a mismatch in the nucleotide sequence.
The allelic association test revealed that allele frequencies were similar in case and control groups (Table 2), while for the rs1801198, in TCN2, marginal values of association were detected. Indeed, the variant allele 776G was more frequent among patients with IPF: the calculated odds ratio was 1.83 (95% C.I. 1.02–3.29; P value= 0.04). However, the permutation analysis by multiple testing correction indicated that this difference was not significant (P value = 0.39). Genotypic association testing provided similar results (Table 3). The rs1801198 polymorphism showed an overall genotypic association close to the nominal levels of significance (P value = 0.06), and an increased risk for the homozygote carrier of the rare allele, OR= 5.25 (95% C.I. 1.19–23.2). The haplotype association analysis, that was conducted for the three polymorphisms of TCN2, supported a genetic association between IPF and TCN2. Indeed, the overall statistics for testing whether any haplotypes were associated, provided evidence of association with threemarker haplotype analysis (P value= 0.04). The specific association values and estimated odds ratio for each of the haplotypes are reported in Table 4. The haplotype 776G (rs1801198)–c.1026-394G (rs7286680)–444C (rs10418) showed a significant association with IPF OR = 2.84 (95% C.I. 1.36–5.93, P value = 0.004), which remained significant after multiple testing correction (P value = 0.04). Discussion IPF is a multistep disease influenced by a variety of cellular alterations. During the first step of IPF, in a restricted pulmonary area,
unknown endogenous and/or environmental stimuli disrupt alveolar epithelial cells, causing abnormal epithelial repair [19]. The alveolar epithelium is normally composed of two types of pneumocytes: type I and type II. Type I are squamous in form and cover the majority of the alveolar surface area (>95%). They are responsible for gas exchange in the alveoli, are unable to replicate, and are susceptible to toxic insults. Type II pneumocytes are the cuboidal and granular cells at the alveolar-septal junction. They cover about 5% of the total alveolar surface area and, in the event of damage, can proliferate and/or differentiate into type I cells. Type II cells are responsible for the production and secretion of surfactant (dipalmitoylphosphatidylcholine), a group of phospholipids that reduce the alveolar surface tension. Several recently-published studies regarding the risk of an association of IPF with SNPs of genes coding MUC5B [16] and the surfactant C and A2 proteins [12,15] revealed genetic bases of IPF linked to a consequent functional alteration of the type II cells which produce these proteins. The main focus of this introductory study (which aims to analyze the SNPs of the folate pathway genes in patients with IPF) is on activated macrophages (another cell type with a key role in IPF) because, in the injured area of alveolar tissue, they express folate receptor β, showing the need for major folate uptake [20]. Cells in rapid division require abundant amounts of folate, essential for the biosynthesis of nucleotides and for methylation [20]. Macrophages continuously clean dirt and microrganisms from the alveolar surface. Cytokines or fragments of pathogenic microbes stimulate the activation of macrophages, giving them enhanced ability to intervene against microrganisms [21]. Activated macrophages also
Table 3 Genotype distribution and overall genotypic association. Gene
Polymorphism ID
CBS MTHFD1 MTHFD1 MTR TCN2 TCN2 TCN2 RFC1 MTHFR DHFR
844ins68 rs2236225 rs1950902 rs1805087 rs1801198 rs7286680 rs10418 rs1051266 rs1801133 rs1643659
Case
Control
Overall assoc.
Heterozygote
Homozygote
AA
Aa
aa
AA
Aa
aa
P value
OR (95% C.I.)
OR (95% C.I.)
29 8 24 25 3 9 23 11 11 11
3 16 7 5 20 17 8 15 15 16
0 7 1 2 9 6 0 6 6 5
73 21 61 50 21 35 56 19 21 35
6 39 19 24 48 36 21 46 42 39
1 20 0 6 12 10 3 15 18 6
0.68 0.95 0.28 0.24 0.06 0.30 0.36 0.49 0.67 0.38
1.26 1.08 0.94 0.42 2.92 1.84 0.93 0.56 0.68 1.31
n.d. 0.92 n.d. 0.67 5.25 2.33 n.d. 0.69 0.64 2.65
(0.29–5.37) (0.40–2.93) (0.35–2.51) (0.14–1.22) (0.78–10.9) (0.72–4.67) (0.36–2.39) (0.22–1.45) (0.27–1.74) (0.53–3.19)
(0.28–3.01) (0.13–3.54) (1.19–23.2) (0.67–8.14) (0.21–2.30) (0.20–2.07) (0.68–10.4)
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Table 4 Haplotype association analysis between TCN2 polymorphisms and IPF. Haplotypes are combination of rs1801198, rs7286680, and rs10418 alleles. Haplotype
C–T-C G–T–C G–G–C G–G–T
Case
Control
Association
n
(%)
n
(%)
P value
OR (95% C.I.)
25 8 21 8
(40) (13) (34) (13)
81 21 24 22
(55) (14) (16) (15)
0.06 0.81 0.004 0.71
1 1.23 (0.49–3.13) 2.84 (1.36–5.93) 1.18 (0.47–2.97)
acquire opposing roles [20,22] depending on the stimulus: in autoimmune diseases (rheumatoid arthritis, lupus, atherosclerosis, psoriasis, diabetes, and transplant rejection) they can cause severe tissue damage. Proinflammatory or profibrotic cytokines produced by them – specifically the macrophage colony-stimulating factor (important for cell proliferation and survival) and TGF-β1 (directly linked to fibrosis) [23] – have higher values in patients with IPF than in patients with other kinds of pulmonary diseases [4,24]. It is believed that macrophages can act on local lung fibroblasts and possibly also recruit fibrocytes [4,25]. An altered proliferation of fibroblasts substituting destroyed pneumocytes causes loss of alveolar function. The same process, occurring in other areas at different times, eventually damages the lungs. The origin of such fibroblasts has not been identified; however, they may be recruited after an epithelial–mesenchymal transition (EMT) [26,27]. It is essential to clarify the role of folate in IPF, given the aberrant proliferation of fibroblasts and particularly of activated macrophages. Although it might seem necessary to prevent folate absorption in order to reduce the proliferation of these cells, it has been proven that cortisone-based drugs, which obstruct folate absorption, do not achieve positive effects. Moreover, cigarette smoking, which also obstructs folate absorption, is itself a risk factor. On the other hand, a therapeutic strategy worth considering could be folate combined with medications. In fact, β receptors of activated macrophages could link with the folate, thereby allowing the other substances in the medication to act at the source of the fibrosis. Our study has a number of limitations. A multicenter study and/or a larger study of an independent patient population would be needed to have an adequate sample size to which to refer. Evaluation of the folate uptake and the folate/B12/homocysteine status of patients with IPF would also be necessary in order to correlate these values with the presence of SNPs in the folate pathway genes. The data obtained from the present investigation indicate no significant differences between patients and control subjects regarding the variant alleles of the SNPs analysed, but the haplotype 776G (rs1801198)–c.1026-394G (rs7286680)–444C (rs10418) for the TCN2 gene showed a significant association with IPF. Our hypothesis that SNPs located on key genes of the folate pathway could exert a possible influence on folate absorption was not confirmed by our results. However, a larger more comprehensive study is required to fully address the question of the real role of SNPs of the folate pathway in IPF. Finally, this introductory study is of significance in order to recognize the importance of further research into the potential therapeutic value of folate–drug conjugates (like those studied for cancer and inflammatory pathologies [22,28,29]) to combat the aberrant proliferation of activated macrophages.
Acknowledgments We are grateful to Dr. Maria Carmen Biffoni and Dr. Daniela Solmi (Laboratorio Analisi Cliniche S. Antonio, Bologna) for their precious collaboration in supplying blood samples from healthy donors. We are indebted to all the patients who agreed to participate in the study.
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