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European Journal of Heart Failure 2007 9(10):1010-1017; doi:10.1016/j.ejheart.2007.07.005
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© 2007 European Society of Cardiology

Promoter polymorphism of the matrix metalloproteinase 3 gene is associated with regurgitation and left ventricular remodelling in mitral valve prolapse patients

Delvac Oceandya,*,1, Rahal Yusoffb,1, Florence M. Baudoina, Ludwig Neysesa and Simon G. Rayb

a Division of Cardiovascular Sciences, University of Manchester Oxford Road, Manchester M13 9PT, United Kingdom
b Department of Cardiology, Wythenshawe Hospital Manchester M23 9LT, United Kingdom

* Corresponding author. 1.302 Stopford Building University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom. Tel.: +44 161 2755672; fax: +44 161 2755669. E-mail address: delvac.oceandy{at}manchester.ac.uk (D. Oceandy).


   Abstract
Top
 Notes
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Background and aims: Mitral valve prolapse (MVP) is common and highly variable in its severity, but the factors underlying this variability are unclear. In this study, we tested the hypothesis that polymorphic variations in Matrix Metalloproteinase (MMP) genes might be predictors of left ventricular (LV) remodelling and severity of regurgitation in MVP.

Methods and results: 70 MVP patients and 75 normal subjects were studied. We performed comprehensive echocardiography and analyzed promoter polymorphisms in the MMP-1 and MMP-3 genes. The MMP-3 -1612 5A/6A polymorphism showed strong associations with indices of mitral regurgitation and LV remodelling: Patients with 5A/5A allele had more pronounced remodelling and more severe mitral regurgitation than patients with the 6A/6A or 5A/6A alleles. We then cloned and sequenced 2 kb fragments of MMP-3 promoter from patients with 5A/5A and 6A/6A genotypes and found 4 different sets of promoter haplotypes. Promoter analysis showed that higher promoter activity was related to a more severe phenotype and that the haplotype variants had a more dominant role in determining the activity.

Conclusions: Our data identifies the MMP-3 promoter haplotype as a novel marker of an adverse disease course in MVP, suggesting the presence of genetic determinants for the severity of MVP.

Key Words: Mitral valve • Genetics • Remodelling • Regurgitation • Echocardiography

Received February 27, 2007; Revised June 14, 2007; Accepted July 11, 2007


   1. Introduction
Top
 Notes
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Mitral valve prolapse (MVP) is a relatively common condition with a prevalence ranging from 2.4% to 15% of the adult population [1,2]. MVP may be a primary abnormality or may occur secondary to other disorders [2]. The exact cause of primary MVP is not completely understood but the contribution of genetic factors is evident. In some family studies, MVP has been linked to chromosomes 16p11.2-p12.1 [3], 11p15.4 [4] and 13q31.3-q32.1 [5]. Others have shown that polymorphisms in several genes are associated with an increased risk of MVP [6-8].

The clinical presentation of MVP varies widely from an asymptomatic murmur to severe mitral regurgitation. Chronic severe mitral regurgitation leads to volume overload of the left ventricle and progressive cardiac remodelling leading in some patients to heart failure. The extent of this adaptive response appears to vary substantially from patient to patient. A number of molecules including the matrix metalloproteinases (MMPs), are known to be involved in determining left ventricular (LV) remodelling during the course of heart failure. MMPs are a family of proteolytic enzymes responsible for extracellular matrix degradation. In the myocardium, several MMPs have been identified including the interstitial collagenase (MMP-1) and stromelysin-1 (MMP-3) [9].

Two single nucleotide polymorphisms ( - 1607 1G/2G and - 1612 5A/6A) have been identified in the promoter region of the MMP-1 and MMP-3 genes respectively and are known to have functional effects on promoter activity [10,11]. Since MMPs play crucial roles in the tissue remodelling process in the heart, it is plausible that these polymorphisms might affect the level of gene expression and hence might influence the LV remodelling process in patients with MVP. To test this hypothesis we analyzed the MMP-1 -1607 1G/2G and the MMP-3 -1612 5A/6A polymorphisms in a population of MVP patients with a variable phenotype in terms of cardiac remodelling and severity of regurgitation.


   2. Methods
Top
 Notes
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Study subjects
We recruited 78 patients with clinically severe mitral regurgitation due to mitral valve prolapse. Patients with any other valvular diseases with the exception of mild tricuspid valve regurgitation were excluded. Patients with ischaemic heart disease, severe heart failure (NYHA IV), uncontrolled hypertension (blood pressure >160/90), renal impairment (creatinine >150 mmol/l), significant respiratory disease and peripheral vascular disease were also excluded. Seventy five control subjects were recruited from the orthopaedic clinics at South Manchester University Hospitals subject to a normal health screening questionnaire and a normal echocardiogram. The study protocol was approved by the local Research Ethics Committee, and all patients and controls gave written informed consent. Symptoms were evaluated using the New York Heart Association Classification.

2.2. Echocardiography
All patients underwent trans-thoracic echocardiography and Doppler examination using a commercially available system (Sonos 5500, Phillips Medical, Eindhoven, Netherlands). Images were recorded on super VHS tapes. Analyses were performed off-line. All measurements were performed according to the American Society of Echocardiography guidelines [12]. Measurements were averaged from three cardiac cycles in sinus rhythm and five cycles in atrial fibrillation. Regurgitant volume and fraction were calculated using the volumetric method [13]. Left ventricular volumes and ejection fraction were calculated using the modified Simpson's biplane method. If the image was sub-optimal in the two chamber view the single plane method from the apical four chamber view was used instead. Left ventricular mass was calculated using the Devereux method [14] and indexed to BSA.

2.3. Polymorphism detection
Genomic DNA was isolated from whole blood using QIAamp DNA blood isolation kit (Qiagen). Polymorphisms were detected by PCR/RFLP (restriction fragment length polymorphism) as described previously [15,16]. Primer sequences used for MMP-1 1G/2G polymorphism were as follows: 5'-TGAGGAAATTGTAGTTAAATCCTTAGAAAG-3' (forward primer) and 5'-TCCCCTTATGGATTCCTGTTTTCTT-3' (reverse primer). Primers for MMP-3 5A/6A polymorphism were: 5'-GATTACAGACATGGGTCACA-3' (forward primer) and 5' TTTCAATCAGGACAAGACGAAGTTT-3' (reverse primer). 100 ng of genomic DNA were subjected to PCR under the condition of initial denaturation for 2 min at 95 °C followed by 30 cycles of 30 s at 95 °C, 30 s at 51 °C and 30 s at 72 °C. The PCR products were digested with the restriction enzyme BslI (New England Biolabs) for MMP-1 1G/2G polymorphism or XmnI (New England Biolabs) for the MMP-3 5A/6A polymorphism and then separated in 2.5% Agarose gel.

2.4. Haplotype analysis
For MMP-3 promoter haplotype analysis, ~2.25 kb MMP-3 promoter fragments flanking the 5A/6A polymorphism site were isolated from patients homozygous for the 5A/5A or 6A/6A genotype by PCR. Primer sequences were as follows: forward: 5'-ATTTACCTGTTTGACATTTGCTATGAGC and reverse: 5'-GGGAGCGCAGCTTTTAAAGAGTGACAGT. PCR products were purified and sequenced to detect the polymorphic variations.

2.5. Promoter activity assay
MMP-3 promoter activity was assayed using luciferase gene as a reporter. MMP-3 promoter fragments were isolated from patients with different haplotypes: 5A-G-A-A-G, 6A-G-C-A-C, 6A-G-C-G-C and 6A-T-C-A-C using PCR as described above, and then cloned into pGL3-basic vector (Promega) which contains luciferase gene. Reporter constructs carrying promoter with ‘synthetic haplotypes’ of 5A-G-C-A-C, 5A-G-C-G-C, 5A-T-C-A-C and 6A-G-A-A-G were also generated.

Promoter activity was analyzed in H9c2 rat ventricular myoblast and NIH3T3 human fibroblast cell lines. 106 cells were cultured in 24-well plates in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) supplemented with 10% Fetal Bovine Serum and 1% penicillin/streptomycin. 1 µg of reporter construct was transfected using Lipofectamine 2000 reagent (Invitrogen) following the manufacturer's recommendation. To control transfection efficiency 0.25 µg of EF-lacZ construct (β-galactosidase under the control of elongation factor promoter) was cotransfected. The MMP-3 promoter activity was examined after 24 h of transfection by measuring the luciferase activity. β-galactosidase activity was also examined for normalisation of transfection efficiency.

2.6. Bioinformatics analysis
MMP-3 promoter sequence (2.5 kb upstream transcription initiation site) was downloaded from Genbank and analyzed using Matinspector (Genomatix Software GmbH) to identify transcription factor binding sites. All analyses were performed using a core similarity >0.7 and an optimized score for matrix similarity, as suggested by Matinspector.

2.7. Statistical analysis
Data are expressed as counts or mean ±SEM. {chi}2 test was used to analyze deviations of genotype distribution from the Hardy-Weinberg equilibrium. The difference in allele frequencies between the MVP and control groups was analyzed using z-test. One way ANOVA was used to analyze associations between genetic polymorphisms and echocardiographic indices. Multiple regression analysis was used to examine whether the associations between polymorphisms and echocardiographic parameters were independent from the effects of covariates such as age, systolic blood pressure, and body surface area. To analyze the promoter activity one way ANOVA followed by post-hoc multiple comparison test was used. Two-tailed values of P<0.05 were considered statistically significant. Statistical analyses were performed using SPSS version 13.0 software.


   3. Results
Top
 Notes
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Patients
Of the 78 MVP patients studied, complete high quality echocardiographic data and DNA for genotyping were available in 70 patients. The mean age was 63.3 years and 70% of patients were male. The clinical characteristics were highly variable in terms of left ventricular remodelling and severity of mitral regurgitation. LV mass ranged from 114 to 527 g (mean value 261 g), and mean mitral regurgitant fraction was 64%.

3.2. Polymorphism detection
The genotype frequencies of MMP-1 1G/2G and MMP-3 5A/6A polymorphisms were tested to evaluate whether there was any deviation from the Hardy-Weinberg equilibrium. Results indicated that the genotype distributions were consistent with the Hardy-Weinberg equilibrium (Table 1). The allele frequencies of each polymorphism were compared with the data obtained from 75 normal subjects and no significant differences were found for the two polymorphisms tested (Table 2), suggesting that the polymorphisms were not related to the occurrence of MVP.


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  		Table 1 {chi}2 test to analyze deviations of genotype distribution from the Hardy-Weinberg equilibrium

 


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  		Table 2 Allele frequency

 
3.3. Associations between polymorphisms and echocardiographic measurements
We examined associations between each polymorphism and the cardiac phenotype of MVP patients obtained from echocardiographic measurements. Echocardiographic data according to the MMP-1 1G/2G and MMP-3 5A/6A genotypes are presented in Table 3. There were no associations between MMP-1 1G/2G polymorphism and echocardiographic indices; however, four parameters of cardiac remodelling and two indices of mitral regurgitation were associated with the 5A/6A MMP-3 polymorphism including LV end diastolic dimension (LVEDD), LV mass, left atrial (LA) volume, mitral annulus diameter, degree of regurgitation and fractional shortening. Post-hoc multiple comparisons tests indicated that patients homozygous for the 5A/5A allele showed significantly greater LVEDD, LV mass, LA volume and mitral annulus diameter while patients with the 5A/6A genotype showed no significant differences compared to patients with the 6A/6A genotype, suggesting that the 5A allele exhibits a recessive effect to the phenotype and that the 5A/5A allele has the detrimental effects on MVP severity.


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  		Table 3 Characteristics of MVP patients according to MMP-1 and MMP-3 genotype

 
Six MVP subjects (8.6%) had atrial fibrillation (AF) and these six subjects were evenly distributed between the three MMP-3 genotypes. Patients with AF had significantly larger left atrial volumes than those without AF but there was no difference in LV mass and dimension (data not shown). It is likely that AF in these patients was secondary to severe mitral regurgitation.

We next analyzed data from the control subjects for associations between these polymorphisms and echocardiographic parameters. Complete echocardiographic data were obtained from 39 control individuals. No significant associations were observed between MMP polymorphisms and parameters of LV remodelling such as LV mass and LVEDD. Two statistically significant results (fractional shortening vs MMP-1 and end systolic volume vs MMP-3) were likely due to statistical artefacts since the differences were in the heterozygote groups (no gene-dosage effect) and the overall values were within the normal range.

Indices of LV remodelling and regurgitation were also compared between male and female patients in each genotype of MMP-3 5A/6A polymorphism. As shown in Table 4, female patients with the 5A/5A genotype displayed a higher regurgitant fraction and lower ejection fraction than male patients with the same genotype raising the possibility that gender might also play a role in determining the cardiac response to MVP.


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  		Table 4 Effects of sex on indices of LV remodelling and regurgitation

 
Multivariate regression analyses were also performed. Factors selected into the multivariate model were age, sex, body surface area, systolic blood pressure and MMP-3 5A/6A polymorphism. The analyses suggested that the 5A/6A MMP-3 polymorphism was an independent predictor of LV mass (P=0.01) and regurgitant fraction (P<0.001) after adjustment for age, body surface area and systolic blood pressure.

3.4. Sequence variation in the MMP-3 promoter
We then followed up our findings by performing further molecular analyses of the MMP-3 promoter. A number of sequence variants have been identified in the promoter region of this gene [17]. Further sequence analysis revealed that the 5A/6A polymorphism was firstly identified in the reverse orientation of the gene [11,18] and therefore the correct nomenclature and position of this polymorphism should be -1612 5T/6T (Genbank sequence accession no.AF405705). However, in this paper we still identify this polymorphism as 5A/6A since this terminology has been widely used. There are four single nucleotide polymorphisms between the –1612 5A/6A polymorphic site and the transcriptional start site (Fig. 1A). Sequence variations were then analyzed in patients homozygous for the 5A or 6A allele and the promoter haplotypes were determined in each patient. Haplotype is a set of closely linked polymorphisms which are inherited as a unit. A combination of sequence variations in the five polymorphic sites within the MMP-3 promoter region constitute the MMP -3 promoter haplotype (Fig. 1A). We found that all patients homozygous for 5A had a similar haplotype: -1612 5A, -1487 G, -1340 A, -707 A, -375 G (this haplotype will be referred as 5A-G-A-A-G in the subsequent text). On the other hand three different haplotypes were found in patients homozygous for 6A, these were: 6A-G-C-A-C, 6A-G-C-G-C and 6A-T-C-A-C (Fig. 1B).


Figure 01
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  		Fig. 1 Polymorphisms at the promoter region of MMP-3 gene A) Schematic diagram of the location of polymorphisms located at the promoter region (2 kb upstream of the transcription start site) of the MMP-3 gene. Sequence number is according to the Genbank sequence accession no.AF405705. B) Haplotype frequency in subjects homozygous of 5A/5A and 6A/6A.

 
3.5. MMP-3 promoter activity
To test whether the 5A/6A polymorphism causes different transcriptional activity of the MMP-3 promoter, we isolated ~2 kb DNA fragments upstream from the transcriptional start site from patients carrying 5A-G-A-A-G, 6A-G-C-A-C, 6A-G-C-G-C and 6A-T-C-A-C haplotypes and generated luciferase reporter constructs. H9c2 ventricular myoblast and NIH3T3 fibroblast cell lines were used to test the promoter activity. In these cells we found that promoters from patients with the 5A-G-A-A-G haplotype displayed significantly increased activity (P<0.01) compared to all promoters with the 6A allele (Fig. 2A). In NIH3T3 cells, promoters with 6A-T-C-A-C had higher activity than promoters with 6A-G-C-A-C and 6A-G-C-G-C haplotypes, whereas in H9c2 cells, promoters with 6A-G-C-G-C had significantly lower activity than promoters with 6A-T-C-A-C and 6A-G-C-A-C haplotypes. This suggests that sequence variations downstream from the 5A/6A polymorphic site are also involved in determining promoter activity.


Figure 02
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  		Fig. 2 Promoter activity assay A) Promoter activity was determined using the luciferase system in H9c2 ventricular myoblast and NIH3T3 fibroblast cell lines. Promoters with the 5A-G-A-A-G haplotype displayed higher activity than all promoters carrying 6A allele (*P<0.01 compared to other promoters; n=10). B) Four synthetic haplotypes (printed in red) were generated to analyze the contribution of 5A/6A polymorphism in determining promoter activity. No significance differences were observed between promoters with the 5A and 6A allele if the downstream haplotype was similar (n=10). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

 
To further investigate whether the 5A/6A polymorphism or the haplotype variants are more dominant in determining promoter activity, we also sub-cloned the G-C-A-C, G-C-G-C and T-C-A-C haplotypes into the 5A construct and the G-A-A-G haplotype into the 6A construct. Results shown in Fig. 2B, indicate that there was no significant difference between promoters with 5A or 6A if the downstream haplotype was similar. These results suggest that the haplotype variants and not the 5A/6A polymorphism are the major determinant of the MMP-3 promoter activity.

Furthermore, potential transcription binding sites in the MMP-3 promoter were predicted using Matinspector software. When the promoter sequence was altered to introduce the polymorphisms, we found considerable changes in transcription binding sites due to the polymorphism at positions –1340 and –375 (Table 5) supporting the data that the 5A/6A polymorphism was not the only major determinant of the MMP-3 promoter activity.


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  		Table 5 Bioinformatics analysis to predict transcription factor binding at the polymorphic sites of MMP-3 promoter

 

   4. Discussion
Top
 Notes
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The MMPs are a family of proteolytic enzymes responsible for extracellular matrix degradation and remodelling. Twenty different types of MMPs have been identified in humans and at least 6 MMPs are expressed in the myocardium including MMP-1, MMP-2, MMP-3, MMP-9, MMP-13 and MMP-14 [9,19]. Myocardial MMP levels are increased during the course of heart failure, in particular during the transition from compensated to decompensated heart failure [20].

Gene polymorphism is any sequence variation within the genome, this can be deletion, insertion or a single base substitution (also called single nucleotide polymorphism = SNP). Polymorphisms that are located at the gene promoter region or regulatory sequence (promoter polymorphisms), have been identified in several MMP genes such as the MMP-1 -1607 1G/2G, MMP-3 -1612 5A/6A, MMP-9 -1562 C/T and MMP-12 -82 A/G. These polymorphisms have been studied in several diseases including ovarian and colorectal cancer, myocardial infarction, aortic aneurysm and coronary atherosclerosis [19]. The MMP-3 5A/6A polymorphism, which has either five or six consecutive adenosine bases, has been associated with high blood pressure [21], acute myocardial infarction [22], aortic stiffening [23] and has been shown as independent predictor of cardiac mortality in patients with non-ischaemic cardiomyopathy [24]. In the present study, we investigated whether polymorphic variations in the promoter of MMP-1 and MMP-3 genes predict the adaptive response to mitral regurgitation in patients with MVP. Our findings indicate that the MMP-1 and MMP-3 polymorphisms did not correlate with the occurrence of MVP, however, the MMP-3 -1612 5A/6A polymorphism appears to be associated with the adversity of MVP, such as LV remodelling and mitral regurgitation. We found that patients homozygous for 5A have greater LV mass and more severe regurgitation than patients with 5A/6A and 6A/6A genotypes. Multiple regression analysis showed that the effect was independent of other covariates such as systolic blood pressure, age, and body surface area. Since the allele frequency of MMP-3 was not different in patients and controls and MMP-3 does not appear to be present in the mitral valve itself [25], there is no reason to suspect that patients in the 5A/5A group are predisposed to more severe mitral valve prolapse per se. However, the 5A/5A polymorphism does appear to influence the pattern of cardiac remodelling in response to the valve lesion, in particular the extent of left ventricular hypertrophy. Regurgitant fraction was significantly greater in patients with the 5A/5A genotype. This was due at least in part to the greater mitral annular diameter in these patients, which may also reflect genetic influences. The greater left atrial volumes are likely to reflect primarily the severity of mitral regurgitation, but could also be genetically mediated.

The next question we addressed was the mechanism by which a polymorphism in the MMP-3 gene might determine the extent of LV remodelling and the severity of regurgitation in MVP patients. Studies by Ye et al. indicated that MMP-3 promoter with the 5A allele has greater activity than the 6A allele [11,17]. Others have shown that the MMP-3 gene transcript in aorta and protein expression in skin are higher in individuals with the 5A/5A than the 5A/6A or 6A/6A genotypes [23]. When we analyzed the activity of the MMP-3 promoter cloned from our MVP patients, we found that the promoter haplotype is a more important determinant of the promoter activity. We identified four major haplotypes in our patient population and generated reporter constructs carrying each promoter haplotype. Our data showed that promoter fragments isolated from patients with the 5A-G-A-A-G haplotype displayed greater activity than promoters with the 6A-G-C-A-C, 6A-G-C-G-C and 6A-T-C-A-C haplotypes. By generating several "synthetic haplotypes" we also demonstrated that the haplotype variation downstream of the 5A/6A polymorphic site and not the 5A/6A polymorphism itself is the determining factor of the promoter activity. In keeping with this finding, bioinformatics analysis revealed that sequence variations at positions –1340 and –375 may also have significant contributions to promoter activity. Although promoter activity assay using luciferase system has some limitations, for example post-translational processing of the luciferase gene might be different from the gene of interest, this system is very sensitive and is able to detect small discrepancies in promoter activity and so it is still the most widely used system for promoter studies.

The present study suggests that MVP patients carrying the more active MMP-3 promoter haplotype have both more severe mitral regurgitation and more pronounced LV remodelling. Evidence that the 5A allele exhibited a recessive effect to the phenotype suggests that two alleles of ‘more active promoter’ might be required to produce the detrimental effect on LV remodelling and regurgitation in MVP. Although we did not measure the levels of cardiac stromelysin directly, we presume that these patients would have higher levels of MMP-3. So what is the possible mechanistic explanation for this finding? The role of MMPs is pivotal in matrix homeostasis and LV remodelling during the course of heart failure. MMP-3 is particularly important because of its broad substrate specificity including collagen, fibronectin, laminin, elastin and proteoglycan [9]. It is known that in volume overload states, such as in mitral regurgitation, myocardial MMP levels are increased [26]. In keeping with this, changes of MMPs and TIMP levels including MMP-3 have been demonstrated in the myocardium in congestive heart failure [27] and hypertensive heart disease [28]. It has also been shown using transgenic models that alterations of MMP levels may disrupt LV remodelling in various pathological models [29,30]. In human heart tissue, MMP-3 expression can be detected in the myocardium [27], but no MMP-3 can be detected in the mitral valve itself using immunohistochemistry [25].

We therefore speculate that in MVP patients with the 5A/5A genotype, increased MMP-3 expression in the myocardium (and not in the mitral valve but possibly in the annulus) accentuates the adaptive response of the heart to the volume overload of mitral regurgitation.

4.1. Study limitations
This study was conducted in a cohort of patients with well-characterized mitral valve prolapse but the number of subjects included is inevitably relatively small. We studied only those patients identified on clinical and echocardiographic grounds as having severe mitral regurgitation and cannot comment on patients with lesser degrees of regurgitation. A potential limitation of this study is our reliance on echocardiography to measure LV size and mass and regurgitant volumes. The echocardiographic methods used for volume and mass calculations are standard and well validated but are less reproducible than measurements obtained by magnetic resonance imaging [31]. We chose to use the quantitative Doppler method to assess MR as we have extensive experience of this method but it is inherently susceptible to errors in the measurement of the areas of the mitral annulus and left ventricular outflow tract. Our findings must therefore be regarded as preliminary and requiring confirmation. Ideally, future studies should use magnetic resonance imaging to assess LV size, mass and morphology to minimize error and increase statistical power.

In conclusion, the present study identifies the MMP-3 promoter haplotype as a novel marker of an adverse disease course in MVP and provides a putative mechanism of action. Clinically, this polymorphism may become one in a matrix of predictors for LV remodelling and regurgitation in MVP patients.


   Acknowledgement
 
This work was supported by Wythenshawe Hospital Cardiac Research Fund.


   Notes
Top
 Notes
Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
1 These authors contributed equally to this work. Back


   References
Top
 Notes
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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