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Journal of the Chinese Medical Association 79 (2016) 656e660 www.jcma-online.com Original Article PRKAG3 polymorphisms associated with sporadic WolffeParkinsoneWhite syndrome among a Taiwanese population

Ken-Pen Weng a,b,*, Yeong-Seng Yuh c, Shih-Hui Huang d, Hsiang-Chiang Hsiao e, Huang-Wei Wu f, Jen-Hung Chien g, Bo-Hau Chen h, Shih-Ming Huang i, Kuang-Jen Chien a, Luo-Ping Ger j,k

a Department of Pediatrics, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan, ROC b Faculty of Medicine, National Yang-Ming University, Taipei, Taiwan, ROC c Department of Pediatrics, Cheng Hsin General Hospital, Taipei, Taiwan, ROC d Department of Nursing, Fooyin University, Kaohsiung, Taiwan, ROC e Division of Cardiology, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan, ROC f Department of Pediatrics, Kaohsiung Armed Forces General Hospital, Kaohsiung, Taiwan, ROC g Department of Pediatrics, Zuoying Branch of Kaohsiung Armed Forces General Hospital, Kaohsiung, Taiwan, ROC h Department of Pediatrics, Taoyuan Armed Forces General Hospital, Taoyuan, Taiwan, ROC i Department of Pediatrics, Kaohsiung Municipal United Hospital, Kaohsiung, Taiwan, ROC j Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan, ROC k Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan, ROC

Received February 15, 2016; accepted August 25, 2016

Abstract

Background: The aim of this study was to investigate whether mutation in AMP-activated protein kinase (AMPK) subunit genes (PRKAG3-230) is associated with sporadic, isolated WolffeParkinsoneWhite (WPW) syndrome. Methods: This study consisted of 87 patients with symptomatic WPW syndrome and 93 healthy controls. PRKAG3-230 genotypes were determined using real-time polymerase chain reaction assay. Genotype and allele frequencies of PRKAG3-230 between patients with WPW syndrome and healthy controls were ascertained using chi-square test or Fisher exact test when appropriate. Results: PRKAG3-230 were genotyped in 87 patients (53 men and 34 women; age ¼ 24.4 ± 18.0 years) with WPW syndrome and 93 healthy controls (57 men and 36 women; age ¼ 16.8 ± 4.2 years). There were no significant differences between the two groups in terms of age and sex. The patients with CG and CGþCC genotypes had a significantly increased risk of WPW syndrome compared with those with GG genotype [odds ratio (OR) ¼ 1.99, 95% confidence interval (CI) ¼ 1.01e3.89, p ¼ 0.045; OR ¼ 1.99, 95% CI ¼ 1.04e3.78, p ¼ 0.037, respectively]. The allelic types were not associated with the risk of WPW syndrome. The patients with manifest type with CG and CGþCC genotypes had a significantly increased risk of WPW syndrome compared with those with GG genotype (OR ¼ 2.86, 95% CI ¼ 1.16e7.05, p ¼ 0.022; OR ¼ 2.84, 95% CI ¼ 1.19e6.80, p ¼ 0.019, respectively). The patients with right-side accessory pathways with CG and CGþCC genotypes had a significantly increased risk of WPW syndrome compared with those with GG genotype (OR ¼ 3.07, 95% CI ¼ 1.25e7.51, p ¼ 0.014; OR ¼ 2.84, 95% CI ¼ 1.19e6.80, p ¼ 0.019, respectively). The allelic types were not associated with the risk of WPW types and locations.

Conflicts of interest: The authors declare that they have no conflicts of interest related to the subject matter or materials discussed in this article. * Corresponding author. Dr. Ken-Pen Weng, Department of Pediatrics, Kaohsiung Veterans General Hospital, Kaohsiung, 386, Ta-Chung 1st Road, Kaohsiung 813, Taiwan, ROC. E-mail address: [email protected] (K.-P. Weng). http://dx.doi.org/10.1016/j.jcma.2016.08.008 1726-4901/Copyright © 2016, the Chinese Medical Association. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). K.-P. Weng et al. / Journal of the Chinese Medical Association 79 (2016) 656e660 657

Conclusion: This study shows that PRKAG3-230 may be associated with sporadic WPW syndrome among a Taiwanese population. Further studies are warranted to elucidate the role of mutations in AMPK subunit genes other than PRKAG3-230 in sporadic WPW syndrome. Copyright © 2016, the Chinese Medical Association. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Keywords: genetics; PRKAG3-230 (rs692243); tachycardia; WolffeParkinsoneWhite syndrome

1. Introduction of >20% in a Han Chinese population. Therefore, SNPs rs692243, rs832678, rs17848621, rs52808491, and The prevalence of WolffeParkinsoneWhite (WPW) syn- rs59655878 were selected for this study. SNPs rs832678, drome based on the electrocardiogram (ECG) criteria is esti- rs17848621, rs52808491, and rs59655878 were not identified mated to be 1e3/1000 individuals.1,2 This syndrome is the in this study because of their complete linkage with rs692243. most common cause of supraventricular arrhythmias in Asian PRKAG3-230 (rs692243) confers a Pro71Ala mutation and is countries and the second most common cause in Western an important variant of R225 mutation in human AMPKg3 countries.3 Most patients with WPW syndrome have struc- subunit.17,18 Costford et al18 reported that R225 mutation has turally normal hearts and are sporadic; however, a minority of the same amino acid location as the mutations in the human g2 cases can be familial4 or involve underlying complicating subunit, which are known to alter AMPK function and cause diseases, such as Ebstein’s anomaly,5 mitochondrial disease,6 WPW syndrome. This led us to investigate whether or not or hypertrophic cardiomyopathy (HCM).7,8 The findings of mutation in PRKAG3-230 is associated with sporadic, isolated Gollob et al8 provided the first strong evidence that mutations WPW syndrome. in the gamma (g)-2 regulatory subunit of AMP-activated protein kinase (AMPK), PRKAG2, are associated with fa- 2. Methods milial HCM and WPW syndrome. The data by Arad et al9 demonstrated that among patients with PRKAG2 mutations, This study consisted of 87 patients (53 men and 34 women; cardiac hypertrophy may be caused due to abnormal glycogen age ¼ 24.4 ± 18.0 years) with symptomatic WPW syndrome storage, resulting in cardiomyocyte vacuolization. The animal and 93 healthy controls (57 men and 36 women; studies further revealed that the annulus fibrosis, which nor- age ¼ 16.8 ± 4.2 years). The patients with symptomatic WPW mally insulates the ventricles from inappropriate excitation by syndrome were recruited from our electrophysiology labora- the atria, was disrupted by glycogen-filled myocytes.10,11 This tory registry of patients with paroxymal supraventricular suggests that ventricular pre-excitations could be related to tachycardia who had undergone electrophysiological study abnormal glycogen storage in cardiac myocytes.10,11 Vaughan from 1990 to 2009. No patient had a family history of WPW et al12 found no evidence that mutations in PRKAG2 cause syndrome or a history of associated hypertrophic cardiomy- sporadic, isolated WPW syndrome; however, they did not opathy. HCM or other cardiac lesion was excluded by two- exclude the contribution of other AMPK subunit genes to dimensional echocardiography (2D-echo) in all patients. We sporadic accessory pathway formation. WPW syndrome is enrolled those healthy controls without a history of arrhythmia reportedly associated with cardiomyopathy in some glycogen and who had normal ECG and 2D-echo. Blood samples were storage disorders, such as Danon and Pompe diseases.13 An collected for PRKAG3-230 analysis after obtaining informed animal study by Milan et al14 demonstrated increased consents from the parents of all participants. This study was glycogen content in the skeletal muscle of Hampshire pigs approved by the Institutional Review Board of Kaohsiung with PRKAG3 mutation, which is similar to human PRKAG2. Veterans General Hospital. For PRKAG3-230 genotyping, Furthermore, Hudson et al15 reported that the glycogen bind- genomic DNA for genotyping was extracted and purified from ing domain of AMPK binds to glycogen and leads to abnormal whole-blood samples of all participants using PUREGENE glycogen-containing inclusions when AMPK is overexpressed. DNA purification kit, (Gentra Systems, Minneapolis, PA, Cheung et al16 demonstrated that PRKAG3, in addition to USA), according to the manufacturer’s instructions. The ge- being expressed in skeletal muscle, is also expressed in the notypes of PRKAG3-230 were detected by the TaqMan real- heart. Their results also suggested that the activity associated time PCR method and subsequently analyzed by ABI with the g3 subunit is very low in some tissue extracts such as PRISM 7500 Sequence Detection System (Applied Bio- heart, but the actual protein expression may be higher.16 It is a systems, Foster City, CA, USA) in 96-well format. Polymerase plausible hypothesis that PRKAG3 mutation may be related to chain reaction (PCR) reactions were performed in reaction glycogen metabolism in the human heart. According to the mixes containing 10 ng DNA, 5 mL 2X TaqMan Universal databases of National Center for Biotechnology Information, PCR Master Mix (Applied Biosystems), 0.5 mL 20X primer/ there are 1140 single-nucleotide polymorphisms (SNPs) in probe mixture, and ddH2O to a final volume of 10 mL. The homo sapiens in the PRKAG3 locus; therefore, we set the PCR program was set as follows: 95C for 10 minutes, fol- selection criteria as missense SNP with minor allele frequency lowed by 40 cycles of 15 seconds at 92C, and 1 minute at 658 K.-P. Weng et al. / Journal of the Chinese Medical Association 79 (2016) 656e660

60C. A single no template control in each 96-well format was Table 2 used for the quality control procedure. The allelic-specific Association of the genotypes with healthy controls and WPW patients. fluorescence data from each plate were analyzed using the Genotypes WPW patients Healthy controls ORa (95% CI) p SDS v1.3.1 software (2005; Applied Biosystems) to auto- (n ¼ 87) (n ¼ 93) matically determine the genotype of each sample. A 5% of No. (%) No. (%) randomly sampled genotypes were repeated for each locus, PRKAG3 and results were 100% consistent with the initial analysis. GG 21 (24.1) 36 (38.7) 1.00 CG 51 (58.6) 44 (47.3) 1.99 (1.01e3.89) 0.045 e 2.1. Statistical analysis CC 15 (17.2) 13 (14.0) 1.98 (0.79 4.95) 0.145 GG 21 (24.1) 36 (38.7) 1.00 CGþCC 66 (75.9) 57 (61.3) 1.99 (1.04e3.78) 0.037 All data are expressed as mean ± standard deviation. De- Values in bold font were statistically significant. mographic data between patients with WPW syndrome and CI ¼ confidence interval; WPW ¼ WolffeParkinsoneWhite. healthy controls were compared using either chi-square test or a Odds ratios and p-values are estimated by logistic regression. 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 were in HardyeWeinberg equilibrium ( p ¼ 0.939). The patients test when appropriate. HardyeWeinberg equilibrium was with CG and CGþCC genotypes had a significantly increased evaluated by comparing the observed to the expected genotype risk of WPW syndrome compared with those with GG genotype frequencies using a goodness of fit chi-square test. The fre- (OR ¼ 1.99, 95% CI ¼ 1.01e3.89, p ¼ 0.045; OR ¼ 1.99, 95% quencies were estimated using SAS/Genetics (Version 9.1.3; CI ¼ 1.04e3.78, p ¼ 0.037, respectively). The allelic types were SAS Institute, Cary, NC, USA) based on the expectation not associated with the risk of WPW syndrome. maximization algorithm. In addition, multiple logistic regres- sion was used to evaluate the association between allele types, 3.2. Genotype and allele frequencies of PRKAG3-230 and genotypes of PRKAG3-230 with risk of WPW syndrome between patients with different WPW types and locations by adjusting various confounders, such as sex and age. Odds and healthy controls ratios (OR) and 95% confidence intervals (CI) were calculated using logistic regression. A p value < 0.05 was considered The genotype and allele frequencies of PRKAG3-230 be- statistically significant. The statistical software package of tween patients with different WPW types and locations are SPSS (Version 17.0; IBM SPSS Inc., Chicago, IL, USA) and shown in Table 3. The genotype distributions of healthy con- SAS/Genetics (Version 9.1.3; SAS Institute Inc., Cary, North trols were in HardyeWeinberg equilibrium ( p ¼ 0.939). The Carolina, USA) were used for all statistical analysis. patients with manifest type with CG and CGþCC genotypes had a significantly increased risk of WPW syndrome 3. Results compared with those with GG genotype (OR ¼ 2.86, 95% CI ¼ 1.16e7.05, p ¼ 0.022; OR ¼ 2.84, 95% CI ¼ 1.19e6.80, PRKAG3-230 were genotyped in 87 patients with WPW p ¼ 0.019, respectively). The patients with right-side acces- syndrome and 93 healthy controls. There were no significant sory pathways with CG and CGþCC genotypes had a differences between the two groups in terms of age and sex significantly increased risk of WPW syndrome compared with (Table 1). those with GG genotype (OR ¼ 3.07, 95% CI ¼ 1.25e7.51, p ¼ 0.014; OR ¼ 2.84, 95% CI ¼ 1.19e6.80, p ¼ 0.019, 3.1. Genotype and allele frequencies of PRKAG3-230 respectively). The allelic types were not associated with the between patients with WPW syndrome and healthy risk of WPW types and locations. controls 4. Discussion The genotype and allele frequencies of PRKAG3-230 be- tween patients with WPW syndrome and healthy controls are Our data suggests that PRKAG3-230 genotypes may be shown in Table 2. The genotype distributions of healthy controls associated with the sporadic WPW syndrome, especially for manifest WPW syndrome and right-side accessory pathways. Table 1 To the best of our knowledge, this is the first study to inves- Comparison of age and sex between healthy controls and WPW patients. tigate the relationship between sporadic WPW syndrome and Factory/category WPW patients Healthy controls P PRKAG3 genetic polymorphisms. (n ¼ 87) (n ¼ 93) Mutations in PRKAG2 were the first to be shown to be 8 No. (%) No. (%) responsible for a familial WPW syndrome . Genetic defects in Age (y), mean ± SD 24.4 ± 18.03 16.8 ± 4.2 0.539 the PRKAG2 gene lead to a diverse cardiac phenotype of Sex variable clinical expressivity and are a rare, autosomal domi- 8 12 Female 34 (39.1) 36 (38.7) nant disease. Vaughan et al analyzed PRKAG2 poly- Male 53 (60.9) 57 (61.3) 0.959 morphism in patients with sporadic WPW syndrome and SD ¼ standard deviation; WPW ¼ WolffeParkinsoneWhite. found that PRKAG2 polymorphism does not predispose to K.-P. Weng et al. / Journal of the Chinese Medical Association 79 (2016) 656e660 659

accessory formation in sporadic WPW syndrome. The mo- a

p lecular 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

44) might have led to the identification of other components of the (95% CI) ¼ Right a n

( kinase complex as new disease genes. It is interesting that 2.84 (1.19-6.80) 0.019 3.07 (1.25-7.51) 0.014 PRKAG3-230 genotypes may be associated with sporadic WPW syndrome in this series. Our findings could have im- plications for other AMP kinase genetic polymorphism other than PRKAG2 responsible for sporadic WPW syndrome. No. (%) OR

87) Experimental studies of familial WPW syndrome associ- ¼

n ated with mutations in PRKAG2 have not completely clarified ( the pathophysiological consequences.10,11,19 Sidhu et al19 re- p ported that a genetic animal model of WPW with excessive

Accessory pathway location cardiac glycogen is due to a loss of function of AMPK. In 10 3.16) 0.340 36 (81.8) 5.54) 0.229 6 (13.6) 2.08 (0.61-7.13) 0.246 3.00) 0.505 30 (68.2) contrast, Arad el al demonstrated that transgenic mutant e e e

43) mice showed elevated AMPK activity and accumulated large 11 ¼ (95% CI) Left amounts of cardiac glycogen. Wolf et al further showed that n a ( 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, 30 (69.8) 1.46 (0.67 21 (48.8) 1.32 (0.58 No. (%) OR 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 13 p Pompe and Danon diseases, can cause WPW syndrome. This suggests that ventricular pre-excitations could be related to microscopic atrioventricular connections rather than conven- 6.80) 0.019 8.90) 0.087 9 (20.9) 1.92 (0.66 7.05) 0.022 e e e tional morphological distinct bypass tracts. A mutation in the 44) g-3 subunit (PRKAG3) associated with excess glycogen ¼ (95% CI) n a ( Manifest content is demonstrated in both animal and human 2.84 (1.19 2.86 (1.16 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 No. (%) OR

87) deposition in WPW syndrome To date, no study has demon- White. ¼ e n strated the unique histologic features of sporadic accessory ( WPW type pathways. However, we postulate that the relationship of p sporadic WPW syndrome with PRKAG3 polymorphism may Parkinson

e be partly explained by the glycogen storage in cardiac myo- 3.16) 0.340 36 (81.8) 4.55) 0.483 8 (18.2) 2.77 (0.86 cytes. Further studies are needed to elucidate the role of e e Wolff PRKAG3-related glycogen storage in sporadic accessory 43) ¼ ¼ (95% CI) pathways. n a ( Concealed 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 eval- uate the relationship of other AMP-kinase subunit genes with odds ratio; WPW

¼ sporadic accessory pathway formation. Furthermore, SNPs in PRKAG3 locus were not completely studied due to the limited

-values are estimated by logistic regression. number of participants. In addition, other genes such as p 93) 20 21 22

¼ endothelin, neuregulin, and T-box2, which have roles in n No. (%) No. (%) OR controls ( 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 confidence interval; OR CC 57 (61.3) 30 (69.8) 1.46 (0.67 Odds ratios and þ WPW syndrome. However, further replication studies should ¼ a Values in bold font wereCI statistically significant. GGCG 36 (38.7) 13 (30.2) 1.00 8 (18.2) 1.00 13 (30.2) 1.00 8 (18.2) 1.00 CC 13 (14.0) 7 (16.3) 1.49 (0.49 Table 3 Association of the genotypes with healthyGenotypes controls, WPW type, and accessory pathway Healthy location. PRKAG3 GGCG 36 (38.7) 44 (47.3) 13 (30.2) 23 (53.5) 1.00 1.45 (0.64-3.26) 0.371 28 (63.6) be conducted 8 (18.2)in 1.00 the future, with large 13 (30.2) cohorts 1.00 in other ethnic 8 (18.2) 1.00 660 K.-P. Weng et al. / Journal of the Chinese Medical Association 79 (2016) 656e660 groups, to more widely demonstrate the applicability of these 10. Arad M, Moskowitz IP, Patel VV, Ahmad F, Perez-Atayde AR, results in other populations. Sawyer DB, et al. Transgenic mice overexpressing mutant PRKAG2 In conclusion, the results of this study show that PRKAG3- define the cause of Wolff-Parkinson-White syndrome in glycogen storage cardiomyopathy. Circulation 2003;107:2850e6. 230 may be associated with sporadic WPW syndrome in a 11. Wolf CM, Arad M, Ahmad F, Sanbe A, Bernstein SA, Toka O, et al. Taiwanese population. Reversibility of PRKAG2 glycogen-storage cardiomyopathy and electro- physiological manifestations. Circulation 2008;117:144e54. Acknowledgments 12. Vaughan CJ, Hom Y, Okin DA, McDermott DA, Lerman BB, Basson CT. Molecular genetic analysis of PRKAG2 in sporadic Wolff-Parkinson- White syndrome. J Cardiovasc Electrophysiol 2003;14:263e8. 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