Heart Vessels (2012) 27:271279 DOI 10.

1007/s00380-011-0152-2

ORIGINAL ARTICLE

Leptin, leptin gene and leptin receptor gene polymorphism in heart failure with preserved ejection fraction
Tarek A. Abd El-Aziz Randa H. Mohamed Rasha H. Mohamed Heba F. Pasha

Received: 5 January 2011 / Accepted: 15 April 2011 / Published online: 17 May 2011 Springer 2011

Abstract Heart failure with a normal ejection fraction (HFNEF) is common in obesity and coronary artery disease (CAD). Both ischemia and reperfusion induce leptin (LEP) and leptin receptor (LEPR) gene expression. We aimed to investigate the possible associations of serum leptin, leptin gene and leptin receptor gene polymorphism with HFNEF in patients with CAD. 100 Egyptian CAD patients with HFNEF and 100 healthy subjects (the control group) were genotyped for LEP and LEPR polymorphism. Leptin levels were measured. Serum leptin levels were signicantly increased in patients compared to the control group. There was a signicant increase in the leptin gene (AA genotype) and the leptin receptor gene (RR genotype) in HFNEF patients compared to the control group. Leptin levels, leptin gene (AA genotype) and LEPR (RR genotype) were more associated with NYHA III than with NYHA I and II. We thus concluded that HFNEF is associated with increased serum leptin levels, and the LEP AA genotype or LEPR RR genotype carries at least a threefold increased risk of developing HFNEF.

Keywords Leptin Leptin receptor Polymorphism Heart failure

Introduction Nearly half of all patients with symptoms of heart failure are found to have a normal left ventricular ejection fraction. Such cases have been termed heart failure with a normal ejection fraction (HFNEF) [1]. In a revealing study, Caruana et al. [2] found that onethird of their patients who had HFNEF were obese, and one half of them showed reduced respiratory function, while many presented evidence of myocardial infarction or ischemia. Although the mechanisms for HFNEF are still not completely understood, diastolic dysfunction is thought to play a role. Another factor that may contribute to HFNEF pathophysiology is abnormal ventriculararterial interaction due to the stiffening of both systems [3]. Leptin has been demonstrated to regulate a variety of cardiac and vascular effects, which include angiogenesis, thrombosis, hemodynamics, and cardiac hypertrophy [4]. Both ischemia and reperfusion induce leptin (LEP) and leptin receptor gene (LEPR) expression [5]. The leptin gene is located on chromosome 7q31.3 and consists of three exons separated by two introns; it expresses a 4.5 kb messenger RNA (mRNA) in adipose tissue [6]. In humans, several polymorphisms have been identied for the LEP G to A substitution at 2548 upstream of the ATG start site in the LEP gene 50 promoter region [7]. Leptin exerts its physiological action through the LEPR. LEPR was initially identied in the brain, which explains its negative feedback mechanism for controlling food intake and body weight [8]. However, the presence of the

T. A. Abd El-Aziz (&) Cardiology Department, Faculty of Medicine, Zagazig University, 28-El-Galaa Street, Zagazig, Egypt e-mail: tarekaziz98@hotmail.com R. H. Mohamed H. F. Pasha Medical Biochemistry Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt e-mail: randahussiny@yahoo.com R. H. Mohamed Biochemistry Department, Faculty of Pharmacy, Zagazig University, Zagazig, Egypt

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leptin receptor in the heart suggests that leptin could modulate cardiac function directly [9]. The human LEPR gene is on chromosome 1p13. A number of polymorphisms have been reported in the human LEPR gene [10]. The LEPR polymorphism Q223R is one of the most common, and is thought to be associated with impaired signaling capacity of the leptin receptor [11]. Circulating levels of leptin have been found to be increased under various pathological conditions, such as obesity [12], chronic obstructive pulmonary disease [13], coronary artery disease (CAD) [14] and chronic heart failure (CHF) [15]. The role of leptin in the etiology or progression of HFNEF is still unclear. Singhal et al. [16] found a marked inuence of leptin on arterial distensibility. Arterial stiffening could inuence diastole by elevating the systolic load to prolong relaxation, compromise lling, and raise left ventricular end-diastolic pressure (LVEDP). We hypothesized that leptin could be a metabolic link between CAD and HFNEF. To the best of our knowledge, there no previous study has addressed the relationship between leptin and HFNEF. The purpose of this study was therefore to clarify the role of leptin, the leptin gene and leptin receptor gene polymorphism in the pathogenesis of HFNEF in patients with CAD.

Medicine, Zagazig University, and written informed consent was obtained from all participants. Biochemical measurements Blood samples were drawn from all subjects after an overnight fast. Total cholesterol and triglycerides were measured by routine enzymatic methods (Spinreact, Girona, Spain). HDL cholesterol was determined after precipitation of the apoB-containing lipoproteins. LDL cholesterol was calculated using the Friedewald formula [17]. Fasting serum leptin concentrations were measured by ELISA (kit provided by Biosource Europe S.A., Nivelles, Belgium) according to Considine et al. [18]. Polymorphism analysis of the G-2548A LEP gene The -2548 G/A polymorphism of the LEP gene was analyzed by the restriction fragment length polymorphism polymerase chain reaction (RFLP-PCR) method described by Mammes et al. [19]. This method was carried out by PCR amplication using the forward primer 50 -TTT CTGTAATTTTCCCGTGAG-30 and the reverse primer 50 -AAAGCAAAGACAGGCATAAAAA-30 in a 25 ll reaction mixture containing the genomic DNA samples (100 ng), 200 lmol/l dNTPs, 1.5 mM MgCl2, 19 Taq polymerase buffer, 50 pmol of each primer, and 0.5 U of Taq DNA polymerase (Amersham, Paris, France). The reaction conditions used with the thermal cycler (Biometra, ttingen, Germany) were as follows: an initial incubation Go at 94C for 5 min, followed by 30 cycles of incubation at 94C for 45 s, 52C for 45 s and 72C for 45 s, with a nal extension at 72C for 7 min. The amplied products were digested with the addition of 2 U CfoI for 3 h at 37C. The digested samples were separated by electrophoresis through a 2% agarose gel and stained with ethidium bromide. The polymorphism was dened by the presence (G) or the absence (A) of the CfoI restriction site. Polymorphism analysis of the Q223R LEPR gene Based on the method described by Gotoda et al. [20], a PCR followed by digestion with the endonuclease MspI was used to detect the A to G transition polymorphism at codon 223 of the LEPR gene. Two sequence-specic oligonucleotide primers were used for the PCR: the 30 primer 50 -AAACTCAACGACACTCTCCTT-30 and the 50 primer 50 -TGAACTGACATTAGAGGTGAC-30 . PCR was performed using 100 ng of genomic DNA as the template, 0.01 mM dNTPs, 1.5 mM MgCl2, 19 Taq polymerase buffer, 0.75 lM of each primer and 0.5 U of Taq DNA polymerase in a total reaction volume of 25 ll. Reaction

Methods We studied 100 Egyptian patients (74 male, 26 female) who underwent cardiac catheterization for evaluation of CAD. Their ages ranged from 37 to 70 years, with a mean of 55.5 8.1. All patients had signicant CAD (at least a 70% stenosis in a major epicardial artery) and LVEDP C 15 mmHg. Patients were excluded if they had one or more of the following: (1) EF \ 50%; (2) acute coronary syndrome, myocardial infarction or stroke within the last 6 months; (3) atrial brillation; (4) chronic obstructive pulmonary disease; (5) severe or life-threatening inammatory illnesses, such as rheumatoid arthritis; (6) cancer; (7) renal failure (serum creatinine [1.4 mg/dl); (8) obesity (body mass index C30). One hundred healthy control Egyptian subjects (72 male, 28 female), who were age- and sex-matched with the HFNEF patients, were randomly recruited. Their ages ranged from 40 to 75 years, with a mean of 50.7 9.5. The control group had no clinical symptoms or signs of CAD, and lacked any family history of premature CAD. They also had no risk factors for atherosclerosis (e.g., diabetes, hypertension, dyslipidemia or obesity). The study was approved by Ethical Committee of the Faculty of

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conditions used with the thermal cycler were as follows: 94C for 5 min, followed by 35 cycles at 94C for 30 s, 52C for 45 s and 72C for 45 s. A nal extension step was carried out at 72C for 5 min. The PCR products (80 bp) were electrophoresed on a 3% agarose gel containing ethidium bromide to monitor amplication and possible contamination. The 80 bp PCR products were digested with MspI and analyzed on 4% agarose gels because of the small size of the DNA products. The presence of the MspI site was indicated by the cleavage of the amplied product into two fragments of 57 and 23 bp. The two allelic forms of LEPR, corresponding to the absence or the presence of the MspI site, are referred to as 223Q and 223R, respectively. Quality control measures included blinded analyses and replicates of 10% of the samples; also, positive controls (blood-derived DNA from all known genotypes) and negative controls for contamination (no DNA) were run routinely with patient samples. Echocardiography A complete echocardiographic examination following a standard protocolincluding a two-dimensional analysis and the assessment of established Doppler parameters that are commonly used to evaluate cardiac diastolic function was performed. All of the examinations and measurements were conducted using a Hewlett Packard (Anaheim, CA, USA) Sonos 5500 with 2.5 MHz transducer. Two-dimensional apical four- and two-chamber views were used to calculate the left atrial volume (LAV) by the biplane area length method and the left ventricular EF using the biplane Simpson method. Cardiac catheterization All patients underwent left heart catheterization through the femoral approach. LVEDP was measured using a 7Fr pigtail uid-lled catheter before angiography. Statistical analysis The results for continuous variables are expressed as the mean SD. The means of the three genotype groups were compared in a one-way analysis of variance. Genotype frequencies in cases and controls were tested for HardyWeinberg equilibrium, and any deviation between the observed and expected frequencies was tested for signicance using the chi-square (v2) test. The correlation coefcients were calculated using Spearman correlation. The statistical signicances of differences in the frequencies of variants between the groups were tested using the v2 test. In addition, the odds ratios (ORs) and

95% CIs were calculated as a measure of the association of the LEP and LEPR genotypes with HFNEF. Multiple regression analysis was performed, adjusted for clinical variables (e.g., age, sex, history of hypertension, history of diabetes, smoking, total cholesterol, triglycerides, HDL, LDL). A difference was considered signicant at P \ 0.05. All data were evaluated using SPSS version 10.0 for Windows.

Results Clinical characteristics of the study subjects Levels of total cholesterol, triglycerides, LDL cholesterol and leptin were signicantly increased in HFNEF patients compared to the control group (Table 1). Furthermore, levels of HDL cholesterol were signicantly decreased in HFNEF patients compared to the control group. LEP and LEPR polymorphisms: genotype and allele distributions In the HFNEF group, the frequency of the LEPR RR genotype was signicantly increased compared to the control group (17% versus 6%, P = 0.007) (Table 2). Subjects with the LEPR-RR genotype were signicantly more likely to develop HFNEF (OR = 3.7, 95% CI = 1.410.3, P = 0.007). The frequency of the R allele was also signicantly increased in the HFNEF group compared to the control group (36 vs 23%, P \ 0.001). Carriers of the R allele were signicantly more likely to develop HFNEF (OR = 8.6, 95% CI = 4.516.2, P \ 0.001). In the HFNEF group, the frequency of the LEP AA genotype was signicantly increased compared to the control group (14 vs. 4%, P = 0.02). Subjects with the LEP AA genotype were signicantly more likely to develop HFNEF (OR = 3.9, 95% CI = 1.212.6, P = 0.02). In NYHA III patients, the frequency of the LEPR RR genotype was signicantly increased compared to NYHA I and II patients (40 vs. 12.5%, P \ 0.001). Subjects with the LEPR-RR genotype were signicantly more likely to have NYHA III (OR = 8.3, 95% CI = 3.718.6, P \ 0.001). Frequencies of the R allele were signicantly increased in NYHA III patients compared to NYHA I and II (60 vs 29.38%), resulting in a signicantly increased OR of subjects bearing the R allele having NYHA III (OR = 3.6, 95% CI = 1.77.3, P \ 0.001). In patients with NYHA III, the frequency of the LEP AA genotype was signicantly increased compared to NYHA I and II (40 vs. 11.25%, P \ 0.001). Subjects with the LEP AA genotype were signicantly more likely to have NYHA III (OR = 7, 95% CI = 3.215.6, P \ 0.001). The frequency of the A allele

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274 Table 1 Clinical, biochemical, and echocardiographic characteristics of the study participants Parameter (n) Age (years) Sex (male/female) Hypertension Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Diabetes Body mass index (kg/m2) Cholesterol (mg/dl) Triglycerides (mg/dl) HDL (mg/dl) LDL (mg/dl) Leptin (ng/ml) Echocardiography Left atrial volume (ml/m2) Ejection fraction (%) Trans-mitral ow Mitral E velocity (cm/s) Mitral A velocity (cm/s) E/A ratio Deceleration time (ms) E/E0 ratio 80.2 8.3 64.6 12.8 1.3 0.096 9.6 1.03 51.3 11.5 73.8 11.1 \0.001 \0.001 \0.001 \0.001 27.2 2.9 68.5 6.4 49.3 20.6 60.3 7.6 \0.001 \0.001 Controls (100) 50.7 9.5 72/28 120 13.8 74 8 25.7 2 185.9 8.4 132 14.4 54.7 3.2 106.2 6.2 15.7 3.3 HFNEF (100) 55.5 8.1 74/26 57 143 22 84 11 41 26.2 1.9 [0.05 240.5 27.4 \0.001 175.7 13.9 \0.001 41.8 4.2 \0.001 163.3 26.6 \0.001 26.1 6.2 \0.001 \0.001 \0.001 Q/Q Q/R R/R n LEPR Q223R 43 27 10 53.75 33.75 12.5 29.38 57.5 31.25 11.25 26.9 4 8 8 24 6 6 8 22 % n P

Heart Vessels (2012) 27:271279 Table 3 Distribution frequencies of the genotypes and alleles of the LEPR and LEP polymorphisms in the patients, as classied according to the NYHA classication Gene NYHA I and II (80) NYHA III (20) % OR 95% CI P

20 40 40 60 30 30 40 55

1 3.2 (1.66.3) 3.6 (1.77.3) 1 0.5 (0.31) 7 3.9 (1.98) 0.06 \0.001 (3.215.6) \0.001 0.001 \0.001 8.3 (3.718.6) \0.001

R allele 47 LEP G2548A G/G G/A A/A 46 25 9

A allele 43

0.67 0.095 \0.001 16.1 1.1

was signicantly increased in the NYHA III group compared to the NYHA I and II groups (55 vs. 26.9%, P \ 0.001), resulting in a signicantly increased OR of subjects bearing the A allele having NYHA III (OR = 3.9, 95% CI = 1.98, P \ 0.001) (Table 3). Association between LEP and LEPR polymorphism genotypes and the clinical characteristics of the study subjects Levels of total cholesterol, triglycerides, and leptin were signicantly higher in LEP AA than in LEP GG (Table 4). Levels of HDL cholesterol were signicantly lower in LEP AA than in LEP GG. Also, levels of total cholesterol, triglycerides, LDL cholesterol and leptin were signicantly higher in LEPR RR than in LEPR QQ. Levels of HDL cholesterol were signicantly lower in LEPR RR than in LEPR QQ. Leptin levels and LVEDP values according to the NYHA classication Leptin levels and LVEDP values were signicantly increased in NYHA III compared to NYHA I and II (Table 5). Association of the study participants characteristics with the severity of HFNEF The difference in NYHA was tested for independence from other variables by multiple regression analysis. The difference in NYHA was found to be dependent on leptin (P = 0.001), triglycerides (P = 0.01), HDL (P = 0.02),

183.7 15.5 255 58.9

E Early diastolic LV lling velocity, A late diastolic LV lling velocity, E0 early diastolic myocardial velocity

Table 2 Distribution frequencies of the genotypes and alleles of the LEPR and LEP polymorphisms in the patients and controls Gene Controls (100) HFNEF (100) OR n LEPR Q223R Q/Q Q/R R/R 60 34 6
,

95% CI

60 34 6 23 56 40 4 24

45 38 17 72 50 36* 14* 64

45 38 17 36 50 36 14 32

1 0.67 0.41.2 3.7 8.6 1 1.01 0.61.8 3.9 1.5 1.212.6 0.92.6 0.9 0.02 0.095 1.410.3 0.2 0.007

R allele 46 LEP G2548A G/G G/A A/A

4.516.2 \0.001

56 40 4*,#

A allele 48

Signicant difference from QQ Signicant difference from QR Signicant difference from GA

*Signicant difference from GG

#

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Heart Vessels (2012) 27:271279 Table 4 Characteristics of the study participants, classied according to the LEPR and LEP genotypes LEPR Q223R Genotype (n) Hypertension, n (%) Diabetes, n (%) Cholesterol (mg/dl) Triglycerides (mg/dl) HDL (mg/dl) LDL (mg/dl) Leptin (ng/ml)

275

LEP G2548A Q/R (72) 31 (43) 19 (26.4) 219 35 162 18 49 6.4 140 35 22.7 5.1 R/R (23) 14 (60.8) 9 (39.1) 239 37 41 6.3
,

Q/Q (105) 29 (27.6) 25 (23.8) 217 34 152 26 47 7.8 139 34 19 5.5

G/G (106) 28 (26.4) 24 (22.6) 217 33 154 25 46 7.3 142 38 18.9 5.5

G/A (76) 38 (50) 21 (27.6) 222 38 161 22 49 7* 140 33 23.6 5.2*

A/A (18) 110 (55.6) 8 (44.4) 233 36* 184 21*,# 41 6.8*,# 155 39 33.7 6.4*,#

185 20,
, ,

161 39

33 5.9,

Signicant difference from QQ Signicant difference from QR Signicant difference from GA

*Signicant difference from GG

#

Table 5 Mean leptin levels and LVEDP in patients classied according to the NYHA classication NYHA I (23) Leptin LVEDP

Association of the study participants characteristics with leptin as well as LEP and LEPR polymorphisms The leptin level was tested for independence from other variables by multiple regression analysis (Table 8). The leptin level was found to be dependent on LEP G2548A polymorphism.

NYHA II (57) 24.4 5.2 20.7 4.7

NYHA III (20) 30.9 7.1 26.4 5.6

26.4 5 17.2 1.9

Signicant difference from NYHA III

and LEP (Table 6).

G2548A

gene

polymorphism

(P = 0.03)

Discussion Association of leptin with HFNEF

Correlation between leptin levels and lipid prole parameters Leptin had signicant positive correlations with total cholesterol, triglycerides and LDL cholesterol, and a signicant negative correlation with HDL cholesterol (Table 7).
Table 6 Association of the study participants characteristics with the severity of HFNEF

The novel nding from this study is that in Egyptian CAD patients who had heart failure with a normal ejection fraction, serum leptin levels were signicantly increased compared to the control group. Moreover, leptin levels and LVEDP were related to exercise intolerance. They showed

Variable

Unstandardized coefcients B SE 0.003 0.08 0.06 0.07 0.06 0.006 0.002 0.008 0.006 0.007 0.08 0.09

Standardized coefcients b 0.055 0.04 -0.025 -0.07 -0.08 0.513 0.301 -0.308 -0.369 0.395 0.029 0.295

95% CI

Age Sex Hypertension Diabetes Smoker Cholesterol Triglycerides HDL LDL Leptin LEPR Q223R LEP G2548A

3.02 9 10-3 3.8 9 10-2 -2.4 9 10-2 -7.8 9 10-2 -7.7 9 10-2 7.4 9 10-3 5.9 9 10-3 -2.1 9 10-2 -5.3 9 10-3 2.7 9 10-2 1.9 9 10-2 0.21

-0.003 to 0.009 -0.12 to 0.2 -0.14 to 0.09 -0.22 to 0.06 -0.2 to 0.05 -0.005 to 0.02 0.001 to 0.01 -0.04 to -0.003 -0.02 to 0.008 0.012 to 0.04 -0.14 to 0.2 0.02 to 0.4

0.97 0.502 -0.42 -1.15 -1.3 1.2 2.6 -2.4 -0.8 3.704 0.25 2.2

0.34 0.61 0.68 0.26 0.2 0.24 0.012 0.02 0.42 0.001 0.803 0.03

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a signicant increase in patients with NYHA III compared to those with NYHA classes I and II. Interestingly, these ndings were associated with signicant increases in the leptin gene (AA genotype) and the leptin receptor gene (RR genotype) in HFNEF patients compared to the control group. We found that subjects carrying LEP AA or LEPR RR had at least a threefold greater risk of developing HFNEF. In addition, the leptin gene (AA genotype) was more associated with NYHA III than with NYHA I and II (OR = 7, P \ 0.001). Also, LEPR (RR genotype) was more associated with NYHA III than with NYHA I and II (OR = 8.3, P \ 0.001). In accordance with our data, Schulze et al. [15] found that patients with CHF have increased serum levels of leptin as compared to healthy controls. Furthermore, patients with CHF and severe exercise intolerance have signicantly higher serum concentrations of leptin than patients with moderate exercise intolerance or healthy controls. Leyva et al. [21] showed that compared to controls who had been matched for both total and regional body fat,

Table 7 Correlations between leptin levels and lipid prole parameters Leptin R Cholesterol Triglycerides HDL LDL 0.361 0.533 -0.46 0.375 P

patients with congestive heart failure are relatively hyperleptinemic. However, in a study by Toth et al. [22], no difference in plasma leptin concentrations was found between patients with congestive heart failure and healthy controls. This variability in results arose because Toth et al.s study included some cachectic patients, whose plasma leptin concentrations tended to be lower than those in noncachectic patients. The results of our study showed that the frequency of the LEPR R allele in the Egyptian population was 23%, which is lower than the gures reported in Japanese (85%), Caucasian (45%), and Pima Indian (32%) [10] populations. With respect to LEP polymorphism, the G allele frequency was higher than the A allele frequency in our study. This is in accordance with the frequencies found in other studies [23, 24], except for one population from Taiwan, where the G allele frequency was somewhat lower than that of the A allele [25]. van der Vleuten et al. [26] found that, in patients with the Q223R polymorphism within the leptin receptor gene, the carriers of one or two R alleles had an increased risk of CHF compared to subjects who were homozygous for the Q allele (OR = 1.6; 95% CI = 1.02.4). It has been reported that there is a relationship between increased leptin levels and progressive functional impairment in advanced CHF, so hyperleptinemia represents a negative prognostic factor in CHF patients [4]. Effect of the G-2548A LEP variant on leptin

\0.001 \0.0001 \0.0001 \0.001

A sequence variation in the gene encoding leptin may affect the expression of this hormone. Although a variation within the coding region of LEP is exceedingly rare in the
LEPR Q223R P 0.4 0.9 0.8 0.9 0.6 0.9 0.6 0.3 0.2 0.8 0.8 0.04 0.6 0.001 Standardized coefcient 0.078 -0.02 -0.004 -0.089 0.103 -0.012 -0.14 -0.03 -0.08 0.03 -0.037 P 0.4 0.8 0.9 0.3 0.3 0.89 0.89 0.75 0.37 0.78 0.78 LEP G2548A Standardized coefcient -0.13 -0.04 -0.06 0.06 -0.05 0.003 0.02 -0.09 -0.04 -0.7 -0.8 0.6 P 0.06 0.6 0.4 0.4 0.4 0.9 0.8 0.3 0.6 0.5 0.04 0.001

Table 8 Association of the study participants characteristics with leptin as well as LEP and LEPR polymorphisms

Variable

Leptin Standardized coefcient

Age Sex Diabetes Smoker NYHA Cholesterol HDL LDL Leptin

-0.12 -0.01 -0.015 0.079 0.001 0.54 -0.2 -0.035

Hypertension -0.04

Triglycerides -0.16

LEPR Q223R -0.09 LEP G2548A -0.8

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general population, several common polymorphisms in the 50 regulatory regionG2548A and A19Gshow associations with LEP levels [27]. Lucantoni et al. [28] showed that the A19G variant present in the untranslated exon 1 of the leptin gene is not associated with any signicant difference in leptin levels. The potential effect of the G-2548A LEP variant on leptin expression has been assessed; however, there have been conicting reports. The present study demonstrates that the leptin level was signicantly higher in LEP AA than in LEP GG. s et al. [19] found In accordance with our data, Mamme that the AA genotype was associated with increased plasma leptin levels in men from a French cohort. This nding was conrmed by a Swedish study, which showed that the -2548A allele of this variant was associated with increased leptin mRNA levels and increased adipose tissue leptin secretion rates [29]. However, in the Taiwanese population, no association was found between this LEP polymorphism and plasma leptin concentrations [25]. Conversely, a relationship between the AA genotype and lower plasma leptin concentrations was described in healthy individuals from Greece, in morbidly obese French patients, and recently in obese Brazilian women [3032]. Effect of LEPR Q223R polymorphism on leptin The relationship between leptin level and the LEPR Q223R polymorphism is unclear. The present study demonstrates that the leptin level was signicantly higher in LEPR RR than in LEPR QQ. In accordance with our data, Yiannakouris et al. [33] found that carriers of the LEPR 223R allele had signicantly higher leptin levels than noncarriers. In contrast, Quinton et al. [11] indicated that the LEPR 223R allele was associated with a lower circulating leptin level. van Rossum et al. [34] demonstrated that R allele carriers have higher leptin levels than people with the homozygotic Q/Q genotype. The LEPR polymorphism leading to a change in charge could considerably impair the ability of leptin to bind to its receptor, and thus provide a phenotype for leptin resistance with inadequate leptin signaling. Other allelic variations in coding and noncoding sequences of the LEPR gene have also been reported, some of which result in silent changes or represent rare mutations [35]. These were not studied here because they are unlikely to have any functional signicance. Association of the LEP and LEPR polymorphisms with the lipid prole In this study we demonstrated that levels of total cholesterol, triglycerides, and leptin were signicantly increased in the LEP AA genotype. Also, levels of total cholesterol,

triglycerides, LDL cholesterol and leptin were signicantly increased in the LEPR RR genotype. Recent studies have reported that the LEPR gene polymorphism inuences serum lipid levels [26, 36]. Patients with the RR genotype had signicantly higher total cholesterol levels and lower high-density lipoprotein cholesterol levels than those with the QQ genotype [37]. Chui et al. [38] have reported that the presence of the R allele correlated with an increased level of total cholesterol, LDL, and the phenomenon of insulin resistance. Leptin or the R allele of the LEPR gene may contribute to low plasma levels of HDL-C by modifying hepatic lipase, phospholipid transfer protein, cholesteryl ester transfer protein, or lipoprotein lipase [39]. Pathophysiological effects of leptin on heart failure Leptin is correlated with markers of inammation such as 9 [15] and CRP [40], which have been associated TNF-a with worse outcomes in patients with CAD [41] and CHF [42]. In the setting of CHF, serum levels of leptin are correlated to altered metabolic, cardiovascular and respiratory parameters, which all contribute to the progression of the underlying disease. While increasing sympathetic nervous outow through the stimulation of hypothalamic receptors, leptin participates in neurohumoral activation during heart failure. The metabolic effects of leptin include increased energy expenditure [22], reduced insulin signaling in muscle [43], impaired fatty acid oxidation, and reduced energy storage. Further, its direct action in the heart involves enhanced levels of reactive oxygen species and prohypertrophic mechanisms, which together increase cardiac energy consumption, attenuate cardiac contractility, and reduce cardiac efciency. Singhal et al. [16] found a marked inuence of leptin on arterial distensibility. The association of arterial distensibility with leptin was independent of fat mass and metabolic and inammatory markers. Arterial stiffness is increased in patients with CAD, and represents an independent predictor of CAD. It has been shown that as the stiffnesses of large arteries such as the aorta increase, cardiovascular morbidity and mortality also increase [44]. In fact, cross-sectional studies have reported a relation between arterial stiffness and LV diastolic function [45]. Arterial stiffness could inuence diastole by elevating the systolic load in order to prolong relaxation, compromise lling, and raise LVEDP [3]. Lieb et al. [46] stated that it is, however, unclear whether leptin contributes to the development of heart failure, or if the higher leptin levels are secondary responses to cardiac damage. The results of our study support the suggestion that leptin contributes to the development of heart failure with normal ejection fraction in CAD patients. This conclusion

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is based on the observation that, in our study, elevated leptin levels in patients with HFNEF are associated with increased frequencies of LEP AA and LEPR RR genotype. Thus, the increase in leptin levels is due to genetic causes, because these genotypes have been shown to be accompanied by increased levels of leptin [19, 29, 33, 34]. Study limitation Our sample may be considered relatively small. Furthermore, the generalizability of our results is limited given the lack of ethnic and racial diversity, because all patients and controls were Egyptians. Lastly, the absence of measurements of fat mass and body fat distribution prevented the correction of leptin levels for total and regional body fat.

Conclusion Heart failure with a normal ejection fraction is associated with an increased serum leptin level. The LEP AA genotype and LEPR RR genotype carries at least a threefold greater risk to develop HFNEF. Increased serum levels of leptin, the LEP AA genotype and the LEPR RR genotype are associated with increased exercise intolerance. Dyslipidemia is associated with increased serum leptin levels, as well as the LEP AA genotype and the LEPR RR genotype.

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