© 2005 The European Society of Cardiology. Published by Elsevier Ltd. All rights reserved.
Angiotensin converting enzyme insertion/deletion polymorphisms in vasovagal syncope
aCardiovascular Investigation Unit (Institute for Ageing and Health), Care of the Elderly Offices, Royal Victoria Infirmary, University of Newcastle Newcastle NE1 4LP, United Kingdom; bSchool of Clinical Medical Sciences, University of Newcastle United Kingdom; cMRC Building, Institute for Ageing and Health, Newcastle General Hospital Westgate Road, Newcastle NE4 6BE, United Kingdom; dInstitute for Human Genetics, International Centre for Life Central Parkway, Newcastle NE1 4BZ, United Kingdom
Manuscript submitted 21 December 2004. Accepted after revision 6 March 2005.
*Corresponding author. Tel.: +44 191 2824128; fax: +44 191 2225638. E-mail address: julianewton{at}blueyonder.co.uk (J.L. Newton).
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Abstract |
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Recent studies suggest vasovagal syncope (VVS) has a significant heritable component (crude estimate sibling relative risk (
s): 1080) indicating that at least some forms of VVS may have a genetic cause. Here we present the first study examining a potential genetic abnormality in VVS. METHODS: DNA was collected from consecutive patients attending our unit with head up tilt confirmed VVS (n = 165). One hundred and fourteen affected and unaffected first-degree relatives of those with a definitive diagnosis of VVS and positive family history also provided DNA.
RESULTS: DNA from 165 VVS index cases was genotyped for the ACE insertion/deletion polymorphism. Mean ± SD age of cases was 56 ± 19 years (103 (62%) females). There was no significant difference in distribution of ACE insertion or deletion gene frequencies in cases compared with a large (>6000 subjects) national control population. No preferential transmission of alleles in families was identified using tests of association (P = 0.1789)
CONCLUSION: We have shown using both a case control and a small family based association study that polymorphisms of ACE alone are not associated with increased risk of VVS. Further studies are planned to clarify the genotype/phenotype relationship in VVS and examine other candidate genes.
Key Words: vasovagal syncope, angiotensin converting enzyme, genetics
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Introduction |
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Vasovagal syncope (VVS) is a common cause of loss of consciousness characterized by hypotension with or without bradycardia, which results in cerebral hypoperfusion and resultant collapse with loss of consciousness. VVS is a disease that clusters in families [1
–3] and recent studies by our group suggest that VVS may have a significant heritable component (crude estimate of sibling relative risk (s): 1080) indicating that at least some forms of VVS may have a genetic cause [4].
A positive diagnosis of VVS is made when a patient has reproduction of symptoms in association with hypotension or bradycardia. Head up tilt test is the investigation carried out to induce these haemodynamic changes [5]. Those with VVS often have low 24 h urinary sodium and previous studies have shown that salt loading increases plasma volume. As a result one form of treatment that reduces symptoms in VVS is dietary salt supplementation [6,7]. Studies carried out by our group suggest that low urinary sodium correlates with symptom severity [8].
As salt supplementation appears to improve orthostatic tolerance and increases baroreceptor sensitivity in those with VVS, any genetic mechanism may reside in abnormalities of sodium handling. The renin angiotensin system is one of the primary mechanisms for salt handling and the angiotensin converting enzyme (ACE, DCP1) insertion (i) deletion (d) polymorphism is frequently invoked as a potential risk factor for hypertension through an action on regulating circulating angiotensin levels [9]. Polymorphisms of the angiotensin converting enzyme would therefore be a plausible potential candidate for regulating some of the features of VVS. Here we present the first study examining a potential genetic abnormality in the common, debilitating condition of VVS.
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Methods |
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The Cardiovascular Investigation Unit (CVIU) in Newcastle sees new referrals from both general practitioners and other hospital units throughout the Northern region of England, an area which shows a relatively stable population structure [10]
. Consecutive individuals with a definitive diagnosis of VVS based on a positive head up tilt test with full symptom reproduction have been identified and all patients have been invited to provide samples of DNA (n = 165). Clinical, haemodynamic and outcome data from all patients with VVS have been collated. Affected and unaffected first degree relatives of those with a definitive diagnosis of VVS and a family history were invited to provide samples of DNA (n = 114), and a comprehensive clinical history of syncopal and presyncopal symptoms was acquired. Genotyping results were compared to a large national control population [9].
DNA preparation and storage
Whole blood (10 ml) was collected from all patients attending the CVIU and family members (as appropriate). Genomic DNA was extracted by standard salting out protocol with chloroform extraction. DNA samples were resuspended and stored at –80 °C. The DCP1 insertion (i) deletion (d) polymorphism in intron 16 was detected using previously described methods and all dd genotypes were conformed using insert specific polymerase chain reaction (PCR) [11]. All reactions were performed in a final volume of 20 µl standard buffer containing 15 pmol of each primer, 0.5 U Taq polymerase (Pharmacia, UK), 200 µM each deoxy-nucleotide, and 200 ng of DNA. Reaction conditions were an initial denaturation at 94 °C for 2 min followed by 35 cycles of annealing at 58 °C for 20 s, extension at 70 °C for 30 s, and denaturation at 94 °C for 30 s. The PCR products were run on a 1% standard: 1% NuSeive agarose (Flowgen, UK) gel, and the bands were visualised using ethidium bromide fluorescence.
Statistical analysis of allele frequencies in the entire VVS cohort was by the Chi-square test with Yates's correction for small sample sizes when appropriate. Where DNA was available from families with at least one affected member with VVS, the transmission disequilibrium test was applied using the program TdTAE [12].
Ethical permission was granted for the study by the Joint Newcastle Ethics Committee and informed consent was obtained from all of the patients enroled in the studies.
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Results |
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Case control study
DNA samples from 165 VVS index cases were genotyped. The mean ± SD years age of this group of cases was 56 ± 19 years with 103 (62%) females. Overall there was no significant difference in the distribution of the ACE insertion or deletion gene frequencies in cases compared with the large (>6000 subjects) national control population (Table 1).
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Thirty-eight of the 165 (23%) of subjects described other family members with syncopal symptoms. DNA was collected from 114 first-degree relatives of the 38 index cases. Using these available families with VVS, no preferential transmission of alleles was identified using TdT based tests of association (Maximum likelihood ratio test (MAX-LRT) 1.808; P = 0.1789).
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Discussion |
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VVS is a common condition, affecting all ages and can profoundly impact upon quality of life. There is emerging evidence that VVS has a strong genetic component and here we present the first candidate gene association study in a cohort of patients with head up tilt diagnosed VVS. Although this study suggests that polymorphisms of ACE alone are not associated with increased risk of VVS, using both a case control and a small family based association study, there may be effects on clinical symptoms.
This study has several limitations. The number of samples is relatively small and family members did not have symptoms confirmed by head up tilt testing. Despite this the differences are such that we are confident that even with a sample size of 10 times that in this study, no effect of ACE polymorphisms would have been detected. A further limitation could be the control population used. Comparing genotyping of the VVS cohort with that of cohorts with unexplained syncope might have been more appropriate, but obtaining a sufficient sample size more difficult, and the national control population used in this study is large and genotyping was performed using the same techniques.
Determining the underlying genetic abnormality in VVS may lead to a better understanding of the pathophysiology of this debilitating condition and result in more appropriate, effective treatment. In addition, a group of people develops VVS symptoms on commencement of vasoactive medication. Determining those with an underlying susceptibility to VVS may allow earlier identification of those at risk of significant side effects. Further studies are planned to clarify the genotype/phenotype relationship in VVS and examine other candidate genes such as serotonin receptor polymorphisms or noradrenaline transporters polymorphisms. Identifying the genes associated with VVS (a hypotensive syndrome) may also have the potential benefit of defining therapeutic targets for hypertension.
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References |
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