Lack of association between genetic polymorphisms affecting sympathetic activity and tilt-induced vasovagal syncope

Autonomic Neuroscience: Basic and Clinical 155 (2010) 98–103

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Autonomic Neuroscience: Basic and Clinical j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / a u t n e u

Lack of association between genetic polymorphisms affecting sympathetic activity and tilt-induced vasovagal syncope Sandro Sorrentino 1, Cinzia Forleo ⁎,1, Massimo Iacoviello, Pietro Guida, Valentina D'Andria, Stefano Favale Cardiology Unit, D.E.T.O. Department, University of Bari, Piazza Giulio Cesare 11, 70124 Bari, Italy

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Article history: Received 8 October 2009 Received in revised form 23 December 2009 Accepted 7 January 2010 Keywords: Vasovagal syncope Tilt table testing Genetic polymorphisms Sympathetic nervous system Italian study

a b s t r a c t Although the pathophysiology of vasovagal syncope is not completely understood, the involvement of sympathetic nervous system alterations has been suggested. Since predisposition to fainting during orthostatic challenge may be associated with genetic variations, we sought to explore the role of genetic polymorphisms affecting sympathetic nervous system function in the susceptibility to tilt-induced vasovagal syncope. We genotyped 129 subjects with recurrent unexplained syncope who underwent tilt testing, and investigated the recurrence of syncope. The analysed polymorphisms were Arg492Cys (ADRA1A gene), Ser49Gly and Arg389Gly (ADRB1), Arg16Gly and Gln27Glu (ADRB2), 825C/T (GNB3), –1021C/T (DBH) and S/L (SLC6A4). No association of the aforementioned genetic variants with both tilt test outcomes and new syncopal episodes during follow-up was found. None of the considered polymorphisms influencing sympathetic activity is a major risk factor for vasovagal syncope in Italian patients. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Vasovagal syncope (VVS) is characterised by a transient failure of the physiological reflexes that contribute to maintaining systemic arterial pressure and cerebral blood flow. However, its largely unknown pathophysiology suggests that studies of its underlying genetic aspects may be useful. In VVS a positive family history has been previously described (Mathias et al., 1998) and recent observations suggest that VVS clusters in families and has a significant heritable component (Màrquez et al., 2005; Newton et al., 2005a). Moreover, a number of genetic polymorphisms have been associated with susceptibility to fainting during orthostatic challenge (Màrquez et al., 2007; Lelonek et al., 2008; Lelonek et al., 2009a; Lelonek et al., 2009b; Saadjian et al., 2009), pointing out sympathetic system involvement in the pathophysiological cascade leading to VVS. These considerations suggest the interest of assessing whether polymorphisms of the factors related to the function of the sympathetic system may predispose to syncope. Post-ganglionic neurons in the sympathetic nervous system principally release norepinephrine as a neurotransmitter that acts through both alphaand beta-adrenergic receptors, and so genes encoding for components of this signal transduction pathway may be involved in the pathogenesis of VVS. ⁎ Corresponding author. Tel.: + 39 080 5478034; fax: + 39 080 5478796. E-mail address: [email protected] (C. Forleo). 1 These two authors contributed equally to this paper. 1566-0702/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.autneu.2010.01.002

The DBH gene encodes dopamine-beta-hydroxylase (DBH), the enzyme catalysing the conversion of dopamine to norepinephrine (Weinshilboum, 1978; Kemper et al., 1987). Plasma DBH activity varies widely among individuals (a subgroup from a large European–American study population was characterised by very low activity levels) (Weinshilboum, 1978), and independently influenced by a number of genetic polymorphisms (Deinum et al., 2004). Zabetian et al. have recently provided strong arguments indicating that an allelic variant in the 5′ flanking region (–1021 C/T) is the major quantitative-trait locus determining plasma DBH concentrations (Zabetian et al., 2001). The α1A adrenergic receptor subtype (α1A-AR) is the predominant α1-AR subtype in the heart and certain parts of the vasculature (Brodde et al., 2001; Kirstein and Insel, 2004; Brodde et al., 2006), and Arg492Cys is a relatively common non-synonymous variant (Xie et al., 1999). Early data indicated that it does not play a functional role (Shibata et al., 1996; Xie et al., 1999; Hsu et al., 2000), but it has been recently suggested that additional in vitro and in vivo studies may be warranted in order to clarify its physiological significance (Snapir et al., 2003; Iacoviello et al., 2006). Numerous functional responses are regulated by β-ARs, including heart rate and contractility, smooth muscle relaxation and multiple metabolic events (Brodde et al., 2001; Kirstein and Insel, 2004; Brodde et al., 2006), and the β1-AR is the predominant subtype in the heart and is also found in other tissues (Brodde et al., 2001; Kirstein and Insel, 2004; Brodde et al., 2006). There are two common single nucleotide polymorphisms (SNPs) in the β1-AR gene (ADRB1) (Brodde et al., 2001; Kirstein and Insel, 2004; Brodde et al., 2006):

S. Sorrentino et al. / Autonomic Neuroscience: Basic and Clinical 155 (2010) 98–103

Ser49Gly and Arg389Gly. The Gly49 variant shows greater baseline receptor activity, greater desensitisation, and is down-regulated much more rapidly after long-term agonist activation than the Ser49 variant (Levin et al., 2002; Rathz et al., 2002), and the Arg389 variant has higher coupling affinity with the Gs-protein than the Gly389 variant, thus leading to 3–4 times greater isoprenaline-stimulated adenyl cyclase activity (Mason et al., 1999). Although β2-ARs are less expressed in the heart than β1-ARs, they are more numerous in many other sites, including the vasculature (Brodde et al., 2001; Kirstein and Insel, 2004; Brodde et al., 2006). There are at least two common SNPs in the β2-AR gene (ADRB2), Arg16Gly and Gln27Glu (Brodde et al., 2001; Kirstein and Insel, 2004; Brodde et al., 2006), the functional consequences of which have been tested as single variations or in the context of specific haplotypes in vitro or in vivo (Brodde et al., 2006). These studies have underlined their influence at the level of agonist-promoted receptor downregulation, with the variation in position 16 playing the dominant role (Brodde et al., 2006). As α- and β-ARs are G protein-coupled receptors, it is expected that polymorphisms in the genes encoding for G proteins may also modulate autonomic nervous system functioning, and it has been reported that the T allele of the 825 C/T polymorphism of the G protein ß3 subunit gene (GNB3) is associated with increased intracellular signal transduction and essential hypertension (Siffert et al., 1998). It is possible that the withdrawal of sympathetic outflow leading to hypotension may be mediated by serotonin, which suggests that the serotoninergic system may be impaired in subjects with VVS (Abboud, 1993). The serotonin transporter encoded by the SLC6A4 gene has a polymorphism in the regulatory region upstream of the coding sequence consisting of a 44 bp insertion or deletion (SLC6A4 L/S) (Heils et al., 1996). In vitro experiments have demonstrated that the long (L) and short (S) variants of this SLC6A4 gene-linked polymorphic region have different transcriptional efficiencies, with the long variant having more basal activity than the short form (Heils et al., 1996; Lesch et al., 1996). We hypothesised that all or any of the genetic polymorphisms affecting sympathetic nervous system functioning may modulate susceptibility to the vasovagal syncope induced by head-up tilt testing (HUT), a major diagnostic means of evaluating patients with neurally mediated syncope (Brignole et al., 2004). We therefore evaluated the role of these genetic polymorphisms in predisposing patients with recurrent unexplained syncope to HUT-induced and subsequent VVS during follow-up. 2. Materials and methods 2.1. Study population We studied 129 consecutive patients from Southern Italy with a history of syncope in the two months preceding enrolment, who were referred to the Syncope Unit of the Institute of Cardiology, University of Bari, between September 2006 and September 2008. The exclusion criteria were an age of less than 18 years; a history of cardiovascular disease, carotid sinus syndrome, or any disease that might affect the autonomic nervous system; and the use of any medication affecting the cardiovascular system. The study was approved by the Ethics Committee of the University of Bari and carried out in accordance with the Declaration of Helsinki; all of the subjects gave their written informed consent. 2.2. Tilt test protocol The head-up tilt test was performed in accordance with the current guidelines (Brignole et al., 2004), as previously described (Iacoviello et al., 2008; Sorrentino et al., 2009).

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Briefly, the tests were performed between 9:00 and 11:00 a.m. in a temperature-controlled room (23 °C) by two nurses experienced in the technique under the supervision of two physicians (C.F. and M.I.). ECG and blood pressure were continuously monitored and recorded using a Task Force Monitor (CNSystems, Graz, Austria). After 10 min of supine rest, the patients were tilted to 70° using an electronically operating tilt-table with a footboard. If VVS had not occurred after 20 min, 300 μg of nitroglycerin was administered sublingually, and the test was continued for a further 20 min. The syncope was classified by two of us (C.F. and M.I.) on the basis of the modified Vasovagal Syncope International Study (VASIS) classification as type 1 (mixed), type 2A (cardioinhibition without asystole), type 2B (cardioinhibition with asystole), or type 3 (vasodepressive). 2.3. Cardiovascular parameters Systolic and diastolic arterial pressure, the RR interval, stroke volume, and total peripheral resistance were measured and analysed off line as previously described (Gratze et al., 1998; Fortin et al., 2006; Iacoviello et al., 2008; Sorrentino et al., 2009). 2.4. Genotyping The –1021 C/T polymorphism of the DBH gene (rs1611115), the Arg492Cys polymorphism of the α1A-AR (rs1048101), the Ser49Gly (rs1801252) and Arg389Gly (rs1801253) polymorphisms of the β1AR, the Arg16Gly (rs1042713) and Gln27Glu (rs1042714) polymorphisms of the β2-AR, the 825 C/T polymorphism (rs5443) of the GNB3 gene, and the L/S polymorphism of the SLC6A4 gene (rs4795541) were characterised as described in previous studies (Heils et al., 1996; Forleo et al., 2004; Iacoviello et al., 2006; Hess et al., 2009; Lelonek et al., 2009a). Genomic DNA was obtained from blood samples using the Wizard Genomic DNA Purification kit (Promega Corporation, Madison, Wisconsin, USA) as recommended by the manufacturer, and was then used for gene amplification by polymerase chain reaction. All of the polymorphisms were amplified using 50 ng of genomic DNA, 40 pm of each primer, and 2 U of AmpliTaqGold polymerase (Roche Molecular Systems, Inc., Branchburg, New Jersey, USA) in a final volume of 25 µl. Fifteen microlitres of the amplified products corresponding to each different polymorphism were digested with the appropriate restriction enzyme following the manufacturer's instructions (New England Biolabs Inc., Ipswich, Massachusetts, USA), and the digestion products were analysed by means of gel electrophoresis using 3% agarose to obtain the genotypes. About 10% of samples with known genotypes were included in each round of genetic screening in order to take in account for genotyping error. In case of discrepancies the analyses were repeated. There were no missing genotypes as all the patients included in the study were successfully screened for the eight polymorphisms. 2.5. Follow-up All of subjects were followed up as outpatients for at least one year, during which any recurrence of syncope was evaluated. Eight patients were lost to follow-up and not included in the analysis concerning the recurrence of syncope. 2.6. Statistical analysis The continuous variables were expressed as mean values ± standard deviation. The groups were compared using the unpaired Student's t test, and frequencies by means of the χ2 or Fisher's exact test. Hardy–Weinberg equilibrium was tested using a χ2 test with one degree of freedom.

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The primary end-point of the study was the association between the genetic polymorphisms and the responses to tilt table testing. Allelic odds ratios (OR) and their 95% confidence interval (CI) for HUT-induced vasovagal syncope were computed by means of a logistic regression analysis. The sample size provided a power >90% to detect an OR>2.5 calculated for the less frequent allele of each polymorphism in HUT+ versus HUT- patients, except for β1-Ser49Gly that showed a power of 68%. The objective of the secondary data analysis was to determine whether the considered polymorphisms were related to syncopal episode occurrence during follow-up. The event-free curves were based on Kaplan–Meier analysis. A Cox proportional hazards model was fitted separately for each of the eight polymorphisms under a dominant model for the less frequent allele. In patients experiencing multiple events, the analysis was restricted to the first event. The results of the Cox proportional hazard analyses are expressed as hazard ratios (HR) and 95% confidence intervals (CI). P values < 0.05 were considered statistically significant. Statistical power was analysed using STATISTICA Power Analysis (StatSoft Inc., Tulsa, Oklahoma), and the statistical analyses were made using STATISTICA 6.1 software (StatSoft Inc., Tulsa, Oklahoma). 3. Results The mean age of the 129 patients enrolled in the study was 35 ± 13 years (range 18–67); 71 (55%) were males. Vasovagal syncope was observed in 73 patients (57%), 28 (22%) during the passive phase of HUT and 45 (35%) after NTG administration; 56 patients (43%) did not faint. Mixed (type 1) syncope was observed in 16 (22%) of the positive patients, cardioinhibitory (type 2A or 2B) syncope in 31 (42%), and vasodepressive (type 3) syncope in 26 (36%). Table 1 shows the genotype and allelic frequency distributions of each polymorphism by tilt test outcome. The frequencies of all of the genotypes were in Hardy–Weinberg equilibrium. There were no significant differences in the genotype or allele frequencies between the two response groups. Clinical variables, cardiovascular parameters and tilt-test outcomes by genetic polymorphisms are displayed in Table 2. The

patients' baseline cardiovascular parameter mean values stratified accordingly to the genotypes were not different. Moreover, the patients were also comparable in terms of age and gender. None of the polymorphisms influenced the risk of having a positive response to tilt-test, nor resulted to be associated with VASIS type of syncope. In Fig. 1 the allelic odds ratios and their 95% confidence intervals estimating the association between the analysed polymorphisms and the susceptibility to vasovagal syncope induced by tilt-test are illustrated. During a mean follow-up of 24 ± 9 months, 25 patients experienced a total of 72 new events (3 ± 4 per patient). The estimated incidence of syncope occurrence over 24 months was 24 ± 4%. Table 3 shows the risks of syncopal events occurred during follow-up of the evaluated patients by genetic polymorphisms. None of the genetic variants was significantly associated with the syncope recurrence. 4. Discussion We studied the involvement of a number of common genetic polymorphisms in predisposing to VVS in a cohort of Italian patients with a history of syncope undergoing HUT. Given the prominent role played by the sympathetic nervous system in VVS pathophysiology, we concentrated on genes that encode components of the system or may be considered as otherwise affecting it on the basis of their functional role. The main end-point of our study was to establish if polymorphisms in these genes could affect HUT outcome in patients with history of VVS. Therefore, we realized the study in patients sharing the same clinical phenotype without reference to a control group of healthy subjects. However, it should be considered that the frequencies of each SNP from each considered gene are not significantly different from those reported in the literature (Brand et al., 1999; Brodde et al., 2001; Kirstein and Insel, 2004; Gerra et al., 2005; Brodde et al., 2006; Haworth et al., 2008; Ross et al., 2008; Hess et al., 2009). The allelic frequencies of SNPs in the adrenergic receptor genes have been extensively reviewed (Brodde et al., 2001; Kirstein and Insel, 2004; Brodde et al., 2006). The allelic frequency of the common ADRA1A

Table 1 Genotype and allele frequency distributions of polymorphisms in subjects with history of syncope. Gene

Polymorphism

Tilt outcome

Genotype 1/1

Genotype 1/2

Genotype 2/2

ADRA1A

Arg492Cys

All Negative Positive All Negative Positive All Negative Positive All Negative Positive All Negative Positive All Negative Positive All Negative Positive All Negative Positive

41 (32) 18 (32) 23 (32) 106 (82) 47 (84) 59 (81) 60 (47) 25 (45) 35 (48) 10 (8) 2 (4) 8 (11) 48 (37) 17 (30) 31 (42) 78 (60) 34 (61) 44 (60) 54 (42) 26 (46) 28 (38) 45 (35) 17 (30) 28 (38)

64 (50) 28 (50) 36 (49) 22 (17) 8 (14) 14 (19) 57 (44) 24 (43) 33 (45) 65 (50) 28 (50) 37 (51) 66 (51) 35 (63) 31 (42) 43 (33) 19 (34) 24 (33) 58 (45) 25 (45) 33 (45) 60 (46) 26 (47) 34 (47)

24 (19) 10 (18) 14 (19) 1 (1) 1 (2) 0 (0) 12 (9) 7 (13) 5 (7) 54 (42) 26 (46) 28 (38) 15 (12) 4 (7) 11 (15) 8 (6) 3 (5) 5 (7) 17 (13) 5 (9) 12 (16) 24 (19) 13 (23) 11 (15)

ADRB1

Ser49Gly

Arg389Gly

ADRB2

Arg16Gly

Gln27Glu

DBH

GNB3

SLC6A4

− 1021 C/T

825 C/T

L/S

P value 0.98

0.65

0.55

0.26

0.07

0.94

0.40

0.42

Allele 1

Allele 2

0.57 0.57 0.56 0.91 0.91 0.90 0.69 0.66 0.71 0.33 0.29 0.36 0.63 0.62 0.64 0.77 0.78 0.77 0.64 0.69 0.61 0.58 0.54 0.62

0.43 0.43 0.44 0.09 0.09 0.10 0.31 0.34 0.29 0.67 0.71 0.64 0.37 0.38 0.36 0.23 0.22 0.23 0.36 0.31 0.39 0.42 0.46 0.38

P value 0.88

0.86

0.44

0.19

0.73

0.85

0.20

0.19

For each polymorphism, 1/1 = homozygosity for the first allele, 1/2 = heterozygosity, and 2/2 = homozygosity for the second allele (e.g. for the Ser49Gly polymorphism: 1/1 = Ser/ Ser, 1/2 = Ser/Gly, and 2/2 = Gly/Gly). OBS1: Genotypes frequencies are given as number of patients (percentage). percentages may not add to 100 because of rounding. ADRA1A: α1A adrenergic receptor; ADRB1: ß1 adrenergic receptor; ADRB2: ß2 adrenergic receptor; DBH: dopamine beta-hydroxylase; GNB3: G protein beta3 subunit; SLC6A4: solute carrier family 6 (neurotransmitter transporter, serotonin) member 4.

Cys

SerSer

917 ± 146 119 ± 12 82 ± 16 1334 ± 283

55 23 32 10 22 23

920 ± 147 117 ± 12 85 ± 17 1284 ± 352

58 20 38 15 27 17

952 ± 158 118 ± 12 86 ± 13 1321 ± 288

61 22 39 13 35 13

55 35 ± 13

55 36 ± 12

61 36 ± 11

60 24 36 13 23 24

912 ± 139 119 ± 12 81 ± 15 1353 ± 335

60 36 ± 13

N = 75

Arg

Gly N = 69

ArgArg N = 60

Gly

Arg389Gly

N = 23

ADRB2 Arg16Gly

ADRB1

GlyGly

52 19 33 11 26 15

928 ± 156 116 ± 10 87 ± 18 1246 ± 282

48 35 ± 13

N = 54

ADRB2

65 23 42 17 25 23

933 ± 155 117 ± 13 81 ± 15 1355 ± 329

63 37 ± 12

N = 48

GlnGln

Gln27Glu T

59 36 ± 13

931 ± 157 118 ± 11 82 ± 16 1345 ± 293

56 24 32 13 26 18

51 34 ± 13

910 ± 140 118 ± 11 85 ± 17 1284 ± 311

52 21 31 10 23 19

57 18 39 12 22 24

899 ± 125 117 ± 13 86 ± 17 1256 ± 349

49 35 ± 12

N = 51

52 22 30 11 24 17

918 ± 147 117 ± 11 84 ± 17 1292 ± 328

59 37 ± 13

N = 54

CC

CC N = 78

Glu N = 81

GNB3 825 C/T

DBH − 1021 C/T T

60 21 39 13 24 23

919 ± 145 118 ± 13 82 ± 16 1324 ± 312

52 34 ± 13

N = 75

SLC6A4

62 22 40 13 20 29

896 ± 144 119 ± 12 80 ± 15 1338 ± 294

53 36 ± 13

N = 45

LL

L/S S

54 21 32 12 26 15

930 ± 146 117 ± 11 85 ± 17 1294 ± 332

56 35 ± 13

N = 84

ADRA1A: α1A adrenergic receptor; ADRB1: ß1 adrenergic receptor; ADRB2: ß2 adrenergic receptor; DBH: dopamine beta-hydroxylase; GNB3: G protein beta3 subunit; SLC6A4: solute carrier family 6 (neurotransmitter transporter, serotonin) member 4. Mean values ± SD. *P < 0.05 versus preceding value within the same group. RRI = RR interval; SAP = systolic arterial pressure; SV = stroke volume; TPR = total peripheral resistance; HUT+: patients fainting before nitroglycerine administration; NTG = nitroglycerin; NTG+: patients fainting after nitroglycerine administration; VASIS: Vasovagal Syncope International Study. OBS2: Percentages were rounded.

56 22 34 12 22 22

912 ± 143 118 ± 12 83 ± 17 1308 ± 325

916 ± 142 118 ± 12 82 ± 15 1315 ± 318

924 ± 155 118 ± 10 86 ± 20 1298 ± 321

57 24 33 11 25 20

54 35 ± 13

N = 106

53 35 ± 12

N = 88

Ser49Gly

ADRB1

59 37 ± 14

Tilt-test outcome (%) Positive 56 HUT+ 17 NTG+ 39 VASIS 1 15 VASIS 2A–2B 22 VASIS 3 20

Baseline RRI (msec) SAP (mm Hg) SV (ml) TPR (dyne*s/cm5)

Males (%) Mean age

N = 41

ArgArg

Arg492Cys

ADRA1A

Table 2 Clinical variables, cardiovascular parameters and tilt-test outcomes by genetic polymorphisms.

S. Sorrentino et al. / Autonomic Neuroscience: Basic and Clinical 155 (2010) 98–103

Arg389Gly

ADRB2

Gln27Glu

Gene Polymorphism Genotypes Reference genotype Hazard ratio (95%CI) P value

ADRA1A Arg492Cys Arg/Arg 0.22

ADRB1 Ser49Gly

1.77 (0.71–4.44) 1.14 (0.43–3.03) 0.54 (0.24–1.22) 1.23 (0.55–2.75) 0.77 (0.35–1.71) 0.97 (0.44–2.17) 1.46 (0.65–3.31) 1.96 (0.78–4.91)

DBH

− 1021 C/T

Cys/Cys + Arg/Cys Gly/Gly + Ser/Gly Gly/Gly + Arg/Gly Arg/Arg + Gly/Arg Glu/Glu + Gln/Glu T/T + C/T

GNB3

825 C/T

T/T + C/T

C/C

SLC6A4

L/S

S/S + L/S

L/L

Arg16Gly

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Fig. 1. Allelic odds ratios for associations between less frequent allele and HUT-induced vasovagal syncope and 95% confidence intervals.

Arg492 variant is 0.54 in Caucasian people (Kirstein and Insel, 2004), which is comparable with the value (0.57) found in our sample. The two SNPs of the ADRB1 gene are in linkage disequilibrium, so that the diplotype Gly49Gly/Gly389Gly occurs very rarely, if at all; the allele frequencies of Gly49 and Gly389 alleles are about 0.12–0.16 and 0.24–0.34, respectively, in Caucasians (Brodde et al., 2006), which are very similar to 0.09 and 0.31 allelic frequencies shown in our study. There is a strong linkage disequilibrium between codon 16 and codon 27 of the ADRB2 gene (Brodde et al., 2006). Thus, subjects homozygous for Glu27Glu are almost always homozygous for Gly16Gly; Arg16Glu27 haplotype occurs naturally very rarely with Arg16Gly and Gln27Glu allele frequencies being 0.38–0.46/0.62–0.54 and 0.65–0.54/0.46–0.35, respectively, in Caucasians (Brodde et al.,

Table 3 Syncope occurrence analysis during follow-up by genetic polymorphisms.

Ser/Ser

Arg/Arg

Gly/Gly

Gln/Gln

C/C 0.80

0.14

0.61

0.52

0.95

0.36

0.15

ADRA1A: α1A adrenergic receptor; ADRB1: ß1 adrenergic receptor; ADRB2: ß2 adrenergic receptor; DBH: dopamine beta-hydroxylase; GNB3: G protein beta3 subunit; SLC6A4: solute carrier family 6 (neurotransmitter transporter, serotonin) member 4. CI = Confidence Interval.

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2006). These data were substantially not different in comparison with those (0.33/0.67 and 0.63/0.37) we have obtained. In addition, we also detected in both ADRB1 and ADRB2 genes the same linkage disequilibrium described in the literature (data not shown), thus confirming that the allelic frequencies of adrenergic receptor SNPs found in our syncopal patient cohort are not significantly different from those reported for control subjects. The C allelic frequency of DBH –1021 C/T polymorphism ranges from 0.78 to 0.83 in four different samples from Caucasian populations (Ross et al., 2008; Hess et al., 2009), very similar to 0.77 of our sample. The L variant of SLC6A4 gene has a frequency of 0.58 in an Italian cohort of control subjects (Gerra et al., 2005) exactly as in our study. Finally, the GNB3 C allele frequency varies between 0.69 and 0.71 in Caucasian populations (Brand et al., 1999; Haworth et al., 2008) versus 0.64 we have observed. In our sample of syncopal patients there was no significant correlation between the considered polymorphisms and the responses to the tilt test. Analysis of the syncopal event recurrence during follow-up by genotypes, that was a secondary end-point of our study, also did not provide significant results. Overall our data suggest that common variants of the DBH, SLC6A4, ADRA1A, ADRB1, ADRB2 and GNB3 genes do not lead to a risk of VVS, at least in the Italian population. Previous studies have examined the association of various genetic polymorphisms with VVS (Newton et al., 2005b; Màrquez et al., 2007; Lelonek et al., 2008; Lelonek et al., 2009a; Lelonek et al., 2009b; Lelonek et al., 2009c; Saadjian et al., 2009; Sorrentino et al., 2009). We have recently shown that the 4A allele of endothelin-1 in the EDN1 3A/4A polymorphism was associated with positive response to tilttest and with vasodepressive pattern of syncope (Sorrentino et al., 2009). A study in Mexican patients found that the Gly389 allele was more frequently present in VVS patients with a positive HUT (Màrquez et al., 2007). Lelonek et al. highlighted that variants of GNAS1, GNB3 and RGS2 genes are associated with predisposition to VVS (Lelonek et al., 2008; Lelonek et al., 2009a; Lelonek et al., 2009b). Moreover, it has been recently shown that in French patients with unexplained syncope, a significant association between high incidence of syncopal episodes, positive HUT, and the presence of the CC variant in the adenosine A2A receptor gene was elicited (Saadjian et al., 2009). However, the current evidence that VVS has a genetic basis is not strong since the number of studies and the amount of data provided is low (Olde Nordkamp et al., 2009). 4.1. Study limitations The risk of syncope arises from a large number of genes, each of which contributes to only a small part of the overall risk, and may be affected by numerous non-genetic factors. Other genetic differences and/or environmental influences in which gene–gene and/or gene– environment interactions have a larger impact on predisposition than the independent effects of each locus may account for the lack of associations in our population. Another important issue is statistical power when small differences in allelic frequencies do not achieve statistical significance. Our sample size was powered to detect only relevant associations between the genotypes and tilt test outcome having a great amount of evidence and identify genetic determinants potentially involved in VVS pathophysiology in patients with history of syncope. In an attempt to avoid confounding factors, we carefully selected the patients on the basis of their history of syncope and the inclusion criteria closely related to those of typical VVS described in the current guidelines. It should also be considered that all the polymorphisms were in Hardy–Weinberg equilibrium, there were no missing genotypes as all the patients included in the study were successfully screened for all the polymorphisms, the genotyping error was accounted by replicating randomly 10% of samples during each

working session and the evaluated population was ethnically homogeneous. These precautions do not exclude the possibility that a bias in our study could be still present due to several factors. In addition, a major limitation of our research is the absence of a replication study in a different population. However, the present should be considered a pilot study suggesting new hypothesis to be tested in prospective studies in different population with several genotyping methods. 4.2. Conclusions To the best of our knowledge, this is the first VVS association study analysing genetic variants of the different components involved in sympathetic system functioning. The results allow us to suggest that none of the considered polymorphisms is a major risk factor for VVS in Italian patients. A better understanding of the genetic determinants of VVS requires further studies of the interactions of the genes involved in the functioning of the sympathetic and parasympathetic systems, and the interactions between gene polymorphisms and environmental factors. Acknowledgements This study was supported by grants from the Italian Ministry of University, Scientific and Technical Research (Nos. ORBA07WPNV and ORBA08N195). The authors are grateful to Ms Margherita Sarlo and Mr. Cataldo Balducci for their excellent technical assistance. References Abboud, F.M., 1993. Neurocardiogenic syncope. N. Engl. J. Med. 328, 1117–1120. Brand, E., Herrmann, S.M., Nicaud, V., Ruidavets, J.B., Evans, A., Arveiler, D., Luc, G., Plouin, P.F., Tiret, L., Cambien, F., 1999. The 825C/T polymorphism of the G-protein subunit ß3 is not related to hypertension. Hypertension 33, 1175–1178. Brignole, M., Alboni, P., Benditt, D.G., Bergfeldt, L., Blanc, J.J., Thomsen, P.E., van Dijk, J.G., Fitzpatrick, A., Hohnloser, S., Janousek, J., Kapoor, W., Kenny, R.A., Kulakowski, P., Masotti, G., Moya, A., Raviele, A., Sutton, R., Theodorakis, G., Ungar, A., Wieling, W., 2004. Task force on syncope, European Society of Cardiology. Guidelines on management (diagnosis and treatment) of syncope — update, Executive summary. Eur. Heart. J. 25, 2054–2072. Brodde, O.E., Bruck, H., Leineweber, K., Seyfarth, T., 2001. Presence, distribution and physiological function of adrenergic and muscarinic receptor subtypes in the human heart. Basic. Res. Cardiol. 96, 528–538. Brodde, O.E., Bruck, H., Leineweber, K., 2006. Cardiac adrenoceptors: physiological and pathophysiological relevance. J. Pharmacol. Sci. 100, 323–337. Deinum, J., Steenbergen-Spanjers, G.C.H., Jansen, M., Boomsma, F., Lenders, J.V.M., van Ittersum, J., Wevers, R.A., 2004. DBH gene variants that cause low plasma dopamine ß hydroxylase with or without a severe orthostatic syndrome. J. Med. Genet. 41, e38. Forleo, C., Resta, N., Sorrentino, S., Guida, P., Manghisi, A., De Luca, V., Romito, R., Iacoviello, M., De Tommasi, E., Troisi, F., Rizzon, B., Guanti, G., Rizzon, P., Pitzalis, M.V., 2004. Association of beta-adrenergic receptor polymorphisms and progression to heart failure in patients with idiopathic dilated cardiomyopathy. Am. J. Med. 117, 451–458. Fortin, J., Habenbacher, W., Heller, A., Hacker, A., Grullenberger, R., Innerhofer, J., Passath, H., Wagner, C., Haitchi, G., Flotzinger, D., Pacher, R., Wach, P., 2006. Noninvasive beat-to-beat cardiac output monitoring by an improved method of transthoracic bioimpedance measurement. Comput. Biol. Med. 36, 1185–1203. Gerra, G., Garofano, L., Zaimovic, A., Moi, G., Branchi, B., Bussandri, M., Brambilla, F., Donnini, C., 2005. Association of the serotonin transporter promoter polymorphism with smoking behavior among adolescents. Am. J. Med. Genet. B Neuropsychiatr. Genet. 135B, 73–78. Gratze, G., Fortin, J., Holler, A., Grasenick, K., Pfurtscheller, G., Wach, P., Schönegger, J., Kotanko, P., Skrabal, F., 1998. A software package for non-invasive, real-time beatto-beat monitoring of stroke volume, blood pressure, total peripheral resistance and for assessment of autonomic function. Comput. Biol. Med. 28, 121–142. Haworth, C.M.A., Butcher, L.M., Docherty, S.J., Wardle, J., Plomin, R., 2008. No evidence for association between BMI and 10 candidate genes at ages 4, 7 and 10 in a large UK sample of twins. BMC Med. Genet. 27, 9–12. Heils, A., Teufel, A., Petri, S., Stober, G., Riederer, P., Bengel, D., Lesch, K.P., 1996. Allelic variation of human serotonin transporter gene expression. J. Neurochem. 66, 2621–2624. Hess, C., Reif, A., Strobel, A., Boreatti-Hummer, A., Heine, M., Lesch, K.P., Jacob, C.P., 2009. A functional dopamine-beta-hydroxylase gene promoter polymorphism is associated with impulsive personality styles, but not with affective disorders. J. Neural. Transm. 116, 121–130.

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