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Original Article

PRKAG3 polymorphisms associated with sporadic Wolff–Parkinson–White syndrome among a Taiwanese population

Weng, Ken-Pena,b,*; Yuh, Yeong-Sengc; Huang, Shih-Huid; Hsiao, Hsiang-Chiange; Wu, Huang-Weif; Chien, Jen-Hungg; Chen, Bo-Hauh; Huang, Shih-Mingi; Chien, Kuang-Jena; Ger, Luo-Pingj,k

Author Information
Journal of the Chinese Medical Association: December 2016 - Volume 79 - Issue 12 - p 656-660
doi: 10.1016/j.jcma.2016.08.008

    Abstract

    1. Introduction

    The prevalence of Wolff–Parkinson–White (WPW) syndrome based on the electrocardiogram (ECG) criteria is estimated to be 1–3/1000 individuals.1,2 This syndrome is the most common cause of supraventricular arrhythmias in Asian countries and the second most common cause in Western countries.3 Most patients with WPW syndrome have structurally normal hearts and are sporadic; however, a minority of cases can be familial4 or involve underlying complicating diseases, such as Ebstein’s anomaly,5 mitochondrial disease,6 or hypertrophic cardiomyopathy (HCM).7,8 The findings of Gollob et al8 provided the first strong evidence that mutations in the gamma (γ)-2 regulatory subunit of AMP-activated protein kinase (AMPK), PRKAG2, are associated with familial HCM and WPW syndrome. The data by Arad et al9 demonstrated that among patients with PRKAG2 mutations, cardiac hypertrophy may be caused due to abnormal glycogen storage, resulting in cardiomyocyte vacuolization. The animal studies further revealed that the annulus fibrosis, which normally insulates the ventricles from inappropriate excitation by the atria, was disrupted by glycogen-filled myocytes.10,11 This suggests that ventricular pre-excitations could be related to abnormal glycogen storage in cardiac myocytes.10,11 Vaughan et al12 found no evidence that mutations in PRKAG2 cause sporadic, isolated WPW syndrome; however, they did not exclude the contribution of other AMPK subunit genes to sporadic accessory pathway formation. WPW syndrome is reportedly associated with cardiomyopathy in some glycogen storage disorders, such as Danon and Pompe diseases.13 An animal study by Milan et al14 demonstrated increased glycogen content in the skeletal muscle of Hampshire pigs with PRKAG3 mutation, which is similar to human PRKAG2. Furthermore, Hudson et al15 reported that the glycogen binding domain of AMPK binds to glycogen and leads to abnormal glycogen-containing inclusions when AMPK is overexpressed. Cheung et al16 demonstrated that PRKAG3, in addition to being expressed in skeletal muscle, is also expressed in the heart. Their results also suggested that the activity associated with the γ3 subunit is very low in some tissue extracts such as heart, but the actual protein expression may be higher.16 It is a plausible hypothesis that PRKAG3 mutation may be related to glycogen metabolism in the human heart. According to the databases of National Center for Biotechnology Information, there are 1140 single-nucleotide polymorphisms (SNPs) in homo sapiens in the PRKAG3 locus; therefore, we set the selection criteria as missense SNP with minor allele frequency of >20% in a Han Chinese population. Therefore, SNPs rs692243, rs832678, rs17848621, rs52808491, and rs59655878 were selected for this study. SNPs rs832678, rs17848621, rs52808491, and rs59655878 were not identified in this study because of their complete linkage with rs692243. PRKAG3-230 (rs692243) confers a Pro71Ala mutation and is an important variant of R225 mutation in human AMPKγ3 subunit.17,18 Costford et al18 reported that R225 mutation has the same amino acid location as the mutations in the human γ2 subunit, which are known to alter AMPK function and cause WPW syndrome. This led us to investigate whether or not mutation in PRKAG3-230 is associated with sporadic, isolated WPW syndrome.

    2. Methods

    This study consisted of 87 patients (53 men and 34 women; age=24.4±18.0 years) with symptomatic WPW syndrome and 93 healthy controls (57 men and 36 women; age=16.8±4.2 years). The patients with symptomatic WPW syndrome were recruited from our electrophysiology laboratory registry of patients with paroxymal supraventricular tachycardia who had undergone electrophysiological study from 1990 to 2009. No patient had a family history of WPW syndrome or a history of associated hypertrophic cardiomyopathy. HCM or other cardiac lesion was excluded by two-dimensional echocardiography (2D-echo) in all patients. We enrolled those healthy controls without a history of arrhythmia and who had normal ECG and 2D-echo. Blood samples were collected for PRKAG3-230 analysis after obtaining informed consents from the parents of all participants. This study was approved by the Institutional Review Board of Kaohsiung Veterans General Hospital. For PRKAG3-230 genotyping, genomic DNA for genotyping was extracted and purified from whole-blood samples of all participants using PUREGENE DNA purification kit, (Gentra Systems, Minneapolis, PA, USA), according to the manufacturer’s instructions. The genotypes of PRKAG3-230 were detected by the TaqMan real-time PCR method and subsequently analyzed by ABI PRISM 7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) in 96-well format. Polymerase chain reaction (PCR) reactions were performed in reaction mixes containing 10 ng DNA, 5 μL 2X TaqMan Universal PCR Master Mix (Applied Biosystems), 0.5 μL 20X primer/probe mixture, and ddH2O to a final volume of 10 μL. The PCR program was set as follows: 95°C for 10 minutes, followed by 40 cycles of 15 seconds at 92°C, and 1 minute at 60°C. A single no template control in each 96-well format was used for the quality control procedure. The allelic-specific fluorescence data from each plate were analyzed using the SDS v1.3.1 software (2005; Applied Biosystems) to automatically determine the genotype of each sample. A 5% of randomly sampled genotypes were repeated for each locus, and results were 100% consistent with the initial analysis.

    2.1. Statistical analysis

    All data are expressed as mean±standard deviation. Demographic data between patients with WPW syndrome and healthy controls were compared using either chi-square test or Student t test. Genotype and allele frequencies of PRKAG3-230 were compared between patients with WPW syndrome and healthy controls using the chi-square test or Fisher exact test when appropriate. Hardy–Weinberg equilibrium was evaluated by comparing the observed to the expected genotype frequencies using a goodness of fit chi-square test. The frequencies were estimated using SAS/Genetics (Version 9.1.3; SAS Institute, Cary, NC, USA) based on the expectation maximization algorithm. In addition, multiple logistic regression was used to evaluate the association between allele types, and genotypes of PRKAG3-230 with risk of WPW syndrome by adjusting various confounders, such as sex and age. Odds ratios (OR) and 95% confidence intervals (CI) were calculated using logistic regression. A p value < 0.05 was considered statistically significant. The statistical software package of SPSS (Version 17.0; IBM SPSS Inc., Chicago, IL, USA) and SAS/Genetics (Version 9.1.3; SAS Institute Inc., Cary, North Carolina, USA) were used for all statistical analysis.

    3. Results

    PRKAG3-230 were genotyped in 87 patients with WPW syndrome and 93 healthy controls. There were no significant differences between the two groups in terms of age and sex (Table 1).

    Table 1
    Table 1:
    Comparison of age and sex between healthy controls and WPW patients.

    3.1. Genotype and allele frequencies of PRKAG3-230 between patients with WPW syndrome and healthy controls

    The genotype and allele frequencies of PRKAG3-230 between patients with WPW syndrome and healthy controls are shown in Table 2. The genotype distributions of healthy controls were in Hardy–Weinberg equilibrium (p=0.939). The patients with CG and CG+CC genotypes had a significantly increased risk of WPW syndrome compared with those with GG genotype (OR=1.99, 95% CI=1.01–3.89, p=0.045; OR=1.99, 95% CI=1.04–3.78, p=0.037, respectively). The allelic types were not associated with the risk of WPW syndrome.

    Table 2
    Table 2:
    Association of the genotypes with healthy controls and WPW patients.

    3.2. Genotype and allele frequencies of PRKAG3-230 between patients with different WPW types and locations and healthy controls

    The genotype and allele frequencies of PRKAG3-230 between patients with different WPW types and locations are shown in Table 3. The genotype distributions of healthy controls were in Hardy–Weinberg equilibrium (p=0.939). The patients with manifest type with CG and CG+CC genotypes had a significantly increased risk of WPW syndrome compared with those with GG genotype (OR=2.86, 95% CI=1.16–7.05, p=0.022; OR=2.84, 95% CI=1.19–6.80, p=0.019, respectively). The patients with right-side accessory pathways with CG and CG+CC genotypes had a significantly increased risk of WPW syndrome compared with those with GG genotype (OR=3.07, 95% CI=1.25–7.51, p=0.014; OR=2.84, 95% CI=1.19–6.80, p=0.019, respectively). The allelic types were not associated with the risk of WPW types and locations.

    Table 3
    Table 3:
    Association of the genotypes with healthy controls, WPW type, and accessory pathway location.

    4. Discussion

    Our data suggests that PRKAG3-230 genotypes may be associated with the sporadic WPW syndrome, especially for manifest WPW syndrome and right-side accessory pathways. To the best of our knowledge, this is the first study to investigate the relationship between sporadic WPW syndrome and PRKAG3 genetic polymorphisms.

    Mutations in PRKAG2 were the first to be shown to be responsible for a familial WPW syndrome8. Genetic defects in the PRKAG2 gene lead to a diverse cardiac phenotype of variable clinical expressivity and are a rare, autosomal dominant disease.8 Vaughan et al12 analyzed PRKAG2 polymorphism in patients with sporadic WPW syndrome and found that PRKAG2 polymorphism does not predispose to accessory formation in sporadic WPW syndrome. The molecular biology of sporadic WPW syndrome seems different from that of familial WPW syndrome. However, they did not exclude the contribution of other AMP kinase subunit genes to sporadic accessory pathway formation.12 Potentially, this might have led to the identification of other components of the kinase complex as new disease genes. It is interesting that PRKAG3-230 genotypes may be associated with sporadic WPW syndrome in this series. Our findings could have implications for other AMP kinase genetic polymorphism other than PRKAG2 responsible for sporadic WPW syndrome.

    Experimental studies of familial WPW syndrome associated with mutations in PRKAG2 have not completely clarified the pathophysiological consequences.10,11,19 Sidhu et al19 reported that a genetic animal model of WPW with excessive cardiac glycogen is due to a loss of function of AMPK. In contrast, Arad el al10 demonstrated that transgenic mutant mice showed elevated AMPK activity and accumulated large amounts of cardiac glycogen. Wolf et al11 further showed that glycogen storage cardiomyopathy and electrophysiological disorders may be attenuated or significantly reversed by the direct modulation of AMPK-mediated cardiac metabolism. These animal studies also revealed that the annulus fibrosis, which normally insulates the ventricles from inappropriate excitation by the atria, was disrupted by glycogen-filled myocytes.10,11 Some glycogen storage disorders, such as Pompe and Danon diseases, can cause WPW syndrome.13 This suggests that ventricular pre-excitations could be related to microscopic atrioventricular connections rather than conventional morphological distinct bypass tracts. A mutation in the γ-3 subunit (PRKAG3) associated with excess glycogen content is demonstrated in both animal and human studies.14,17,18 PRKAG3, in addition to being expressed in skeletal muscle, is also expressed in the heart and is related to glycogen storage.16 Considerable interest may arise regarding the possible mechanism to explain the role of gylcogen deposition in WPW syndrome To date, no study has demonstrated the unique histologic features of sporadic accessory pathways. However, we postulate that the relationship of sporadic WPW syndrome with PRKAG3 polymorphism may be partly explained by the glycogen storage in cardiac myocytes. Further studies are needed to elucidate the role of PRKAG3-related glycogen storage in sporadic accessory pathways.

    Some limitations of our study need to be considered. Our study was a single-center investigation comprising a limited number of participants. Additionally, our study did not evaluate the relationship of other AMP-kinase subunit genes with sporadic accessory pathway formation. Furthermore, SNPs in PRKAG3 locus were not completely studied due to the limited number of participants. In addition, other genes such as endothelin,20 neuregulin,21 and T-box2,22 which have roles in the development of the cardiac conduction system and the atrioventricular ring, may be attractive candidate genes to be evaluated as contributors to the pathogenesis of sporadic WPW syndrome. However, further replication studies should be conducted in the future, with large cohorts in other ethnic groups, to more widely demonstrate the applicability of these results in other populations.

    In conclusion, the results of this study show that PRKAG3-230 may be associated with sporadic WPW syndrome in a Taiwanese population.

    Acknowledgments

    This research was supported in part by the Kaohsiung Veterans General Hospital (VGHKS100-086, VGHUST104-G7-7-3, VGHKS105-106, VGHUST105-G3-1-3).

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    Keywords:

    genetics; PRKAG3-230 (rs692243); tachycardia; Wolff–Parkinson–White syndrome

    © 2016 by Lippincott Williams & Wilkins, Inc.