Advance Publication

Circulation Journal ORIGINAL ARTICLE doi: 10.1253/circj.CJ-19-0415 Arrhythmia/Electrophysiology

Rs4968309 in Light Chain 4 (MYL4) Associated With Atrial Fibrillation Onset and Predicts Clinical Outcomes After Catheter Ablation in Atrial Fibrillation Patients Without Structural Heart Disease

Yuan Zhong, MD; Kai Tang, MD, PhD; Hailing Li, MD, PhD; Dongdong Zhao, MD, PhD; Wenxin Kou, MD; Shaojie Xu, MD; Jun Zhang, MD; Haotian Yang, MD; Shuang Li, MD, PhD; Rong Guo, MD, PhD; Wenhui Peng, MD, PhD; Yawei Xu, MD, PhD

Background: Atrial fibrillation (AF) is the most common arrhythmia with serious complications and a high rate of recurrence after catheter ablation. Recently, mutation of MYL4 was reported as responsible for familial atrial cardiomyopathy and AF. This study aimed to determine the association between polymorphism in MYL4 with the onset and recurrence of AF.

Methods and Results: A total of 7 single-nucleotide polymorphisms were selected by linkage disequilibrium and genotyped in 510 consecutive AF patients and 192 controls without structural heart disease. A total of 246 AF patients who underwent cryoballoon ablation had a 1-year scheduled follow-up study for AF recurrence. C allele and CC genotype of rs4968309 and A allele of rs1515751were associated with AF onset both before and after adjustment of covariation (age, sex, hypertension, and diabetes). AF type and left atrial size were different among the genotypes of rs4968309. Moreover, CC genotype of rs4968309 increased susceptibly of AF recurrence after cryoballoon ablation. The prevalence of hypertension was associated with rs1515752, and left atrial size was associated with the genotype of rs2071438.

Conclusions: C allele and CC genotype of rs4968309 in MYL4 were associated with AF onset and recurrence. Moreover, the A allele of rs1515751 had a significant association with AF onset. The polymorphisms of MYL4 can predict AF onset and prognosis after ablation in AF patients without structural heart disease.

Key Words: Atrial fibrillation; Catheter ablation; MYL4; Single-nucleotide polymorphisms

trial fibrillation (AF) is the most common arrhythmia the gap junction a 1 (GJA1) ,13 a homozygous affecting an estimated 1–2% of the population and mutation in the nucleoporin 155 (NUP155) gene,13 and the is associated with serious outcomes such as stroke natriuretic peptide A (NPPA) gene.14 A 1,2 and . Although catheter ablation is an Recently, using exome sequencing and systematic bioin- effective method of improving life span and quality of life formatics analyses, we identified a rare missense variant for many AF patients, there are still 20–40% of patients of the gene encoding essential type-4 who require further management.3 AF is associated with (MYL4 c.31G>A.p.E11K), causing missense-induced several clinical risk factors, such as diabetes mellitus, loss-of-function in a large multiplex atrial cardiomyopathy obesity, and hypertension, and its development is affected family pedigree.15 Two recent studies have also discussed by genetic background. To date, rare variants of more than the significance of variants in MYL4 in familial atrial 30 have been identified as correlating with AF.4–6 cardiomyopathy and AF.16,17 The MYL4 protein forms a Most of these studies have focused on ion channels, including collar around the lever of the myosin heavy chain, close to cardiac sodium, calcium, and potassium.6–12 Linkage and the site forming -myosin cross-bridges, and regulates candidate gene studies have also identified rare genetic the interaction.18 This 21.6-kDa protein consisting of 197 variants of non-ion channel-related genes in individuals amino acids virtually disappears from the human with familial or lone AF, such as a frameshift mutation in after birth, becomes essentially atrial-specific, and reappears

Received May 13, 2019; revised manuscript received June 19, 2019; accepted July 2, 2019; J-STAGE Advance Publication released online August 10, 2019 Time for primary review: 15 days Department of Cardiology, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai, China ORCID of Hailing LI: 0000-0002-9903-3683 The first two authors contributed equally to this work (Y.Z., K.T.). Mailing address: Yawei Xu, MD, PhD and Hailing Li, MD, PhD, Department of Cardiology, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, 301 Yanchang Road, Shanghai 200072, China. E-mail: [email protected]; [email protected] ISSN-1346-9843 All rights are reserved to the Japanese Circulation Society. For permissions, please e-mail: [email protected] Advance Publication 2 ZHONG Y et al. at the ventricular level in response to ventricular pathology.18 cardioversion, either with drugs or by direct current The corresponding ventricular protein, MYL3, is essential cardioversion. Coronary artery disease was defined as the for normal ventricular function. MYL3 mutations cause occurrence of exercise- and stress-related chest symptoms hypertrophic cardiomyopathy.19 Our further study indi- caused by ≥70% narrowing of 1 or more of the major cated that loss-of-function MYL4 variants cause progressive coronary arteries and ≥50% in the left main coronary atrial cardiomyopathy in humans and rats, and MYL4 is a artery.24 The LAd and left ventricular key gene required for atrial contractile, electrical, and (LVEF) were measured by transthoracic ultrasonic testing. structural integrity.15 Heart failure patients were New York Heart Association Monogenic mutations in lone and familial AF, although class IV25 or LVEF <40%.26 Body mass index (BMI) was rare, have been recognized as both gain- and loss-of- calculated as weight divided by height squared. Hyperten- function mutations in the same gene and can cause AF.20 sion was defined as blood pressure >140/90 mmHg or the Genome-wide association studies have indicated that use of antihypertensive medication. Diabetes was diagnosed common single-nucleotide polymorphisms (SNPs) have a according to the WHO criteria. Cryoballoon ablation for role in the development of AF. Common genetic variants 246 AF patients was performed by qualified physicians using associated with AF may be adopted in the clinical setting, a standard ablation procedure that has been previously such as risk stratification of the development of AF, esti- reported in detail.27 mation of the efficacy of pulmonary vein isolation, and indication of anticoagulant therapy. Excepting the rare gene SNPs Selection and DNA Genotyping variants, SNPs associated with AF may identify individuals DNA was extracted from peripheral blood using standard at an increased risk and enable stratification of ablation phenol chloroform extraction techniques. The 192 control therapy or peri-interventional management. There has been cohort and 288 AF patients were randomly selected for a study based on large-scale gene sequencing of Icelanders direct sequencing of MYL4. Genotyping was performed in that identified SNPs (rs185183057, rs28588212, rs117626672, the remaining the samples with TaqMan allelic discrimi- rs117626672) in MYL4 that predisposed to AF.21,22 In this nation by means of an ABI 7900HT (Applied Biosystems, study, we scanned all sites of MYL4 by gene sequencing in Foster City, CA, USA). The TaqMan assay kits and small Chinese cohorts with and without AF and found a probes were obtained from Applied Biosystems. All PCR new SNP rs4968309 differing significantly in the AF and data were analyzed using ABI Prism SDS software version non-AF cohorts. Next, we selected 6 SNPs in MYL4 2.3. The samples that PCR failed to detect or distinguish according to linkage disequilibrium via Haploview and production were excluded. Based on the results of sequencing, genotyping for all 7 SNPs to expand the participation. we found that rs4968309 differed significantly in the AF Through baseline data analysis and follow-up of patients and non-AF cohorts. Next, we selected 6 SNPs in MYL4 after catheter ablation, we investigated whether those SNPs according to linkage disequilibrium via Haploview. were associated with AF onset or recurrence after ablation in AF patients without structural heart disease. Follow-up Study All participants were monitored by qualified physicians Methods during their hospital stay and the procedure (i.e., the first 24–48 h) with ECGs and Holter studies. For patients who Study Design and Subjects’ Characteristics underwent cryoballoon ablation there were scheduled Our study complied with the Declaration of Helsinki and visits at 1, 3, 6, and 12 months after discharge, or earlier was approved by the hospital’s ethical review board if symptoms were consistent. 24-h Holter and 12-lead (Shanghai Tenth People’s Hospital, Tongji University ECG were necessary to determine the rapid recurrence of School of Medicine, Shanghai, China). Written informed atrial arrhythmias. Recurrence of AF was defined as any consent was given by all subjects before their participation recording of AF on ECG or an episode >30 s on 24-h in the study. This was an observational study of 510 Holter after a blanking period of 3 months or a repeat consecutive AF patients from the Department of Cardiology, ablation at any time. Atrial tachycardia was defined as a Shanghai Tenth People’s Hospital who were diagnosed regular, organized supraventricular rhythm at a constant with non-valvular AF according to the diagnostic criteria atrial rate >100 beats/min. Based on the morphology and of the AF guidelines23 between March 2016 and May 2018. regularity of the rhythm, early and late recurrences were The 192 non-AF controls were patients who were admitted classified as AF or AT. to the Department of Cardiology at the same time and met the exclusion criteria. Exclusion criteria included: (1) acute Statistical Analysis or chronic infections or inflammatory diseases, severe Statistical Package for Social Sciences (SPSS) for Windows liver or renal dysfunction, malignant tumor, hematologic 10 was used for statistical analysis. Unpaired t-test and disorders, and history of cerebral infarction or transient Chi-square test were used for continuous traits and cate- cerebral ischemia within 6 months; (2) myocarditis or gorical traits respectively. For the allelic analysis, Chi-square cardiomyopathies; (3) reversible AF caused by acute alco- test and Fisher’s exact test were used to compare the holism, cardiopulmonary surgery, endocrine abnormalities frequency of SNP variation between AF and non-AF etc.; (4) recent interventional treatment of acute coronary patients and then confirmed in 3 genetic models. To further events; (5) undergone left atrial (LA) ablation or cardiac evaluate the relationship between alleles and AF, alleles surgery; (6) LA diameter (LAd) >60 mm; and (7) heart with P<0.05 in the Chi-square test or Fisher’s exact test failure. were selected and multiple logistic regression analysis was Paroxysmal AF was defined as AF that terminated performed with risk factors of AF (age, sex, hypertension, spontaneously or with intervention within 7 days of onset. and diabetes mellitus) as adjusted covariates. For the Persistent AF was defined as continuous AF sustained ≥7 predictors of AF recurrence after cryoballoon ablation, days, or episodes beyond >7 days that were terminated by survival analysis was performed to test the effect of the risk Advance Publication SNPs in MYL4 Associated With AF 3

Table 1. Demographic and Basic Clinical Characteristics of the Study Population Non-AF AF Non-recurrence Recurrence Characteristic P value P value (n=192) (n=510) (n=197) (n=49) Age (M ± SD, years) 60.15±7.53 65.66±9.10 <0.001 65.35±9.79 63.70±7.24 0.278 Sex (male, %) 79 (41.80) 250 (56.43) 0.001 116 (58.59) 26 (55.32) 0.683 BMI (M ± SD, kg/m2) 24.06±3.08 25.52±3.61 0.012 25.07±3.19 26.45±2.85 0.268 Hypertension (n, %) 6 (3.16) 265 (60.64) <0.001 105 (54.40) 27 (58.70) 0.599 DM (n, %) 2 (1.04) 70 (16.02) <0.001 25 (12.95) 9 (19.57) 0.249 Thyroid dysfunction (n, %) 1 (0.53) 5 (1.15) 0.464 4 (2.07) 0 (0.00) 0.325 pro-BNP (M ± SD, pg/mL) 90.21±142.40 791.56±1,524.57 <0.001 572.32±835.80 562.85±480.33 0.943 LDL (M ± SD, mg/dL) 107.12±31.71 106.73±274.94 0.977 88.94±31.71 90.10±29.78 0.841 AF type (persistent/paroxysmal) NA 111/326 NA 23/170 17/29 <0.001 LVEF (M ± SD, %) 60.81±18.41 58.69±13.82 0.106 59.03±13.42 59.80±14.10 0.730 LAd (M ± SD, mm) 35.58±4.55 42. 43±6.47 <0.001 40.52±5.40 43.15±5.99 0.004 Data are mean±standard deviation (M ± SD), unless otherwise shown. AF, atrial fibrillation; BMI, body mass index; BNP, B-type natriuretic peptide; DM, diabetes mellitus; LAd, left atrial diameter; LDL, low-density lipoprotein; LVEF, left ventricular ejection fraction; NA, not applicable.

allele using Kaplan-Meier test. Odds ratio (OR) and 95% confidence interval (CI) are reported. Numerical variables with a normal distribution are presented as the mean ± SE, while numerical variables with a skewed distribution are presented as the median, and categorical variables are presented as percentages. Two-tailed P<0.05 was considered statistically significant for all analyses. In addition to multiple comparisons, the P-value was corrected to <0.01 based on Bonferroni adjustment in post hoc analysis. According to the minor allele frequency (MAF) ranging from 0.112 for rs4968309 to 0.465 for rs16941662 reported in HapMap of the East Asian population, OR of the study was 1.5, promising >80% statistical power to detect an association (at P<0.05) under the dominant model.

Results Subjects’ Characteristics There were 510 AF patients and 192 non-AF controls in our study, including 246 AF patients who underwent cryoballoon ablation. In the AF cohort, 25.40% was persistent AF and 74.60% was paroxysmal AF. With regard to general demographic characteristics (age, sex and BMI) Figure 1. Positions of the single-nucleotide polymorphisms and basic clinical factors (hypertension, diabetes mellitus, (SNPs) in the MYL4 gene and linkage disequilibrium plot (LD) LAd and pro-BNP), there were statistical differences calculated with Haploview software and shown in the red between the AF and control groups (Table 1). AF patients diamonds. had larger LAd and more prevalence of hypertension and diabetes mellitus compared with non-AF patients (P<0.001 for both). In addition, AF patients had high BMI and increased pro-BNP. However, thyroid dysfunction, LVEF, Rs4968309 and rs1515751 Associated With the Risk of AF and serum level of low-density lipoprotein (LDL) were not Onset significantly different between the AF and non-AF groups. According to the results of direct sequencing, the frequency We followed the 246 AF patients who underwent of the minor C allele in rs4968309 was significantly higher cryoballoon ablation, including 21 patients who relapsed in AF cases. For the selected 6 SNPs (rs1515751, rs2071438, within 3 months (early recurrence of AF, ERAF) and 28 rs16491971, rs16491662, rs151572, and rs9894365) with R2 patients with late recurrence of AF (LRAF, between 3 and of linkage disequilibrium exceeding 0.8 compared with 12 months). In total, 19.51% of the AF patients relapsed in rs4968309 by Haploview (Figure 1), the MAF of all SNPs our follow-up study. As shown in Table 1, compared with exceeded 0.05 and was similar to the frequencies in the the non-recurrence cohort, there were more patients Chinese population reported by the Hapmap Project. diagnosed as persistent AF among the cases of relapses Table 2 shows the genotype and allele frequencies of the (11.92% vs. 42.5%, P<0.001), and the recurrence group had MYL4 polymorphisms, with the ancestral allele of each larger LAd compared with the non-recurrence cohort SNP used as the referent for minor allelic homozygote (43.47±5.61 vs. 40.51±5.46 mm, P=0.02). and heterozygote analyses. Rs4968309 and rs1515751 had different frequencies in AF patients and non-AF controls (P<0.001 and P=0.003, respectively). After Bonferroni Advance Publication 4 ZHONG Y et al.

Table 2. Genotype Distribution in Controls and AF Patients SNPs / Control AF OR Control AF OR P value Allele P value Genotype (n=192) (n=510) (95% CI) (n=192) (n=510) (95% CI) rs4968309 TT 134 383 Ref. T 309 797 0.388 1.163 (74.44) (80.63) (85.83) (83.89) (0.825, 1.639) CT 41 31 <0.001 3.780 C 51 153 (22.78) (6.53) (2.278, 6.272) (14.17) (16.11) CC 5 61 0.001 4.268 (2.78) (12.84) (1.680, 10.848) rs2071438 AA 133 369 Ref. A 303 813 0.524 0.876 (78.24) (82.00) (89.12) (90.33) (0.584, 1.316) AG 37 75 0.162 0.731 G 37 87 (21.76) (16.67) (0.470, 1.135) (10.88) (9.67) GG 0 6 0.317 NA (0.00) (1.33) rs1515751 GG 151 204 Ref. G 310 442 0.001 3.507 (94.97) (84.65) (97.48) (91.70) (1.619, 7.595) AG 8 34 0.003 3.146 A 8 40 (5.03) (14.11) (1.416, 6.990) (2.52) (8.30) AA 0 3 0.369 NA (0.00) (1.24) rs1515752 TT 84 349 Ref. T 229 787 <0.001 0.443 (56.38) (78.25) (76.85) (88.23) (0.316, 0.621) TG 61 89 <0.001 0.351 G 69 105 (40.94) (19.96) (0.235, 0.526) (23.15) (11.77) GG 4 8 0.408 0.481 (2.68) (1.79) (0.142, 1.636) rs9894365 GG 80 226 Ref. G 209 634 0.848 0.973 (52.98) (48.92) (69.21) (68.61) (0.734, 1.289) GA 49 182 0.185 0.761 A 93 290 (32.45) (39.39) (0.507, 1.141) (30.79) (31.39) AA 22 54 0.621 1.151 (14.57) (11.69) (0.659, 2.010) rs16941662 TT 42 128 Ref. T 164 486 0.818 0.970 (26.92) (27.29) (52.56) (51.81) (0.751, 1.254) CT 80 230 0.791 1.060 C 148 452 (51.28) (49.04) (0.689, 1.632) (47.44) (48.19) CC 34 111 0.795 0.934 (21.80) (23.67) (0.556, 1.568) rs16941671 CC 66 323 Ref. C 199 765 <0.001 0.564 (47.83) (69.31) (72.10) (82.08) (0.413, 0.771) GC 67 119 <0.001 0.363 G 77 167 (48.55) (25.54) (0.243, 0.541) (27.90) (17.92) GG 5 24 0.970 0.981 (3.62) (5.15) (0.361, 2.664) Genotype distribution of SNPs between groups: rs4968309, P<0.001; rs2071438, P=0.119; rs1515751, P=0.005; rs1515752, P<0.001; rs9894365, P=0.273; rs16941662, P=0.860; rs16941671, P<0.001. CI, confidence interval; OR, odds ratio; SNP, single-nucleotide polymorphism.

correction, the difference was still significant for P<0.01. P=0.001, OR=6.368, respectively). An allele of rs1515751 As shown in Table 3, under additive, dominant, and was associated with AF onset under the dominant model recessive models, the C allele CC genotype of rs4968309 (P=0.001, OR=4.441), and the association remained signifi- (P=0.001, OR=4.268 under additive model and P=0.001, cant after covariation adjustment (P=0.001, OR=6.022). OR=5.157 under recessive model) was associated with AF onset. Multivariable analysis with covariations (age, sex, Rs4968309 and rs2071438 Associated With LAd in AF hypertension, and diabetes) showed that the CC genotype Patients of rs4968309 was still associated with AF onset under both Clinical data for participants with different genotypes are the additive and recessive models (P=0.002, OR=5.397 and shown in Table 4. Both LV function and LAd were within Advance Publication SNPs in MYL4 Associated With AF 5

Table 3. Analysis of rs4968309 and rs1515751 Under Additive, Dominant and Recessive Models OR Exp(b) SNPs P value P-adj (95% CI) (95% CI) rs4968309 Additive CC 0.001 4.268 (1.680, 10.848) 0.002 5.397 (1.845, 15.785) TT Dominant CC+CT 0.083 0.700 (0.467, 1.049) 0.197 0.698 (0.404, 1.206) TT Recessive CC <0.001 5.157 (2.037, 13.054) 0.001 6.368 (2.192, 18.502) TT+CT rs1515751 Additive AA 0.369 NA NA NA GG Dominant AA+AG <0.001 4.441 (1.825, 10.810) 0.001 6.022 (1.995, 18.176) GG Recessive AA 0.412 NA NA NA GG+AG Covariations: age, sex, hypertension and diabetes. Exp(b), adjusted OR; P-adj, adjusted P value. Other abbreviations as in Tables 1,2.

Table 4. Demographic and Basic Clinical Characteristics of AF and Non-AF Patients Among the Genotype of SNPs rs4968309 rs2071438 rs1515751 rs1515752 rs9894365 rs16941662 rs16941671 (n=655) (n=620) (n=400) (n=598) (n=613) (n=625) (n=604) Characteristic CC/CT/TT GG/AG/AA AA/AG/GG GG/TG/TT AA/GA/GG CC/TC/TT GG/GC/CC (66/72/517) (6/112/502) (3/42/355) (12/150/436) (76/231/306) (145/310/170) (29/186/389) Sex (male, n) 29/34/246 1/52/230 0/19/151 7/68/207 35/119/136 66/152/78 15/89/184 Age (M±SD, years) 63.87±8.97 64.17±8.80 63.66±9.55 63.86±8.94 64.06±9.03 63.96±9.07 64.20±8.99 BMI (M±SD, kg/m2) 25.10±3.53 25.12±3.49 24.62±3.23 25.09±3.52 25.12±3.55 25.04±3.55 25.11±3.58 DM 9/7/48 0/17/43 0/5/18 2/13/48 10/25/29 21/30/15 5/19/41 Hypertension 29/22/202 2/41/194 0/16/84 3/49/189* 33/103/111 65/121/65 16/73/158 AF type Non-AF 5/41/134 0/37/133 0/8/151 4/61/84 22/49/80 34/80/42 5/67/66 Paroxysmal AF 44/20/231* 2/56/224 1/24/129 3/62/213 36/117/138 73/147/78 14/73/203 Persistent AF 6/11/91* 2/11/84 0/3/13 3/19/80 13/45/46 29/52/23 10/32/63 LAd (M±SD, mm) Referent 40.87±6.68* 40.93±6.83* 38.84±5.87 41.22±6.71 40.80±6.70 41.06±6.71 41.20±6.67 Alternation 41.13±6.55 41.00±8.28 40.33±11.59 42.25±6.54 41.47±7.75 41.55±7.22 44.69±7.86 Heterozygote 38.12±7.47* 39.04±6.30* 39.90±5.73 39.65±7.24 40.78±6.68 40.25±6.61 39.99±6.60 LVEF (M±SD, %) Referent 59.07±15.45 58.87±15.60 60.70±13.40 58.79±15.68 59.12±15.99 60.22±15.50 58.69±15.47 Alternation 57.05±15.67 49.50±24.36 60.67±1.16 66.50±7.68 58.38±15.91 59.11±14.86 53.45±20.65 Heterozygote 60.35±17.98 60.24±14.32 60.03±10.37 57.92±17.82 59.37±14.55 58.59±15.70 60.14±14.54 pro-BNP (M ± SD, pg/mL) Referent 618.30± 635.06± 380.31± 663.21± 625.61± 421.08± 713.07± 1,467.93 1,498.71 835.72 1,543.15 1,725.48 693.71 1,675.26 Alternation 723.51± 773.83± 406.198± 434.55± 425.66± 620.55± 676.84± 1,120.84 505.75 878.46 713.84 642.12 1,057.04 845.65 Heterozygote 284.27± 471.35± 630.28± 482.29± 639.14± 658.60± 466.03± 404.37 859.17 1,179.41 932.30 1,042.68 1,649.56 799.54 Data are mean ± standard deviation (M ± SD), unless otherwise shown. *P<0.01. Abbreviations as in Tables 1,2.

normal ranges among the AF and non-AF patients. Geno- The prevalence of hypertension was associated with the G types of rs4968309 were significantly associated with LAd allele of rs1515752 under the dominant model (P=0.001). and AF type distribution (P=0.002 and P=0.028). The CC genotype of rs4968309 was persistent with AF (P=0.019, Variations of rs4968309 Associated With AF Recurrence OR=2.889 (1.190, 7.012)). LAd was also associated with In the univariate analysis, rs4968309 was associated with rs2071438 under the dominant model (P=0.011, Figure 2). AF recurrence (P=0.040). Indeed, both the univariable Advance Publication 6 ZHONG Y et al.

Figure 2. Association of rs4968309 (A) and rs2071438 (B) with left atrial size in the AF and non-AF patients. AF, atrial fibrillation.

in maintaining isometric tension.29 MYL4 is expressed in Table 5. Multivariable Analysis of rs4968309 and AF Recurrence Under Recessive Model both atria and ventricles in embryonic life; however, after birth, ventricular MYL4 expression plummets and the Variations P value Exp(b) (95% CI) protein becomes highly atrial selective.28 Forced ventricular Sex 0.132 1.850 (0.832, 4.116) overexpression of MYL4 in transgenic rats enhances the Age 0.127 0.963 (0.917, 1.011) muscle shortening velocity, rate of tension development, DM 0.093 2.548 (0.857, 7.576) and isometric force production,30 possibly explaining the Hypertension 0.734 1.154 (0.506, 2.633) teleological role of MYL4 re-expression in diseased human LAd 0.007 1.116 (1.030, 1.209) ventricles.29–31 MYL4 overexpression protects against LVEF 0.380 1.020 (0.976, 1.066) contractile impairment in volume-overloaded rats.31 LDL 0.683 1.115 (0.660, 1.884) Atrial standstill, a complete loss of atrial electrical and pro-BNP 0.863 1.000 (0.999, 1.001) mechanical function, eventually develops in many patients rs4968309 recessive 0.017 6.709 (1.411, 31.904) with MYL4 mutations; in some, mechanical function is greatly reduced before atrial electrical activity is lost.15 Exp(b), adjusted OR. Abbreviations as in Tables 1,2. Several lines of evidence suggest that the MYL4 E11K mutation is not directly responsible for atrial standstill, but rather that it results from the delayed consequences of analysis and multiple regression analysis with adjustment atrial remodeling induced by the mutation. First, the hypo- for sex, age, diabetes, hypertension, LVEF, LAd, LDL, and contractile effects of the mutation are generally expressed pro-BNP demonstrated that patients with the CC genotype at birth, whereas atrial standstill develops much later in of rs4968309 had increased susceptibly to AF recurrence life. Second, MYL4 dysfunction reduces atrial contractility (P=0.044 and P-adj=0.017 for the recessive model, Table 5). but does not necessarily eliminate it. Third, knockout of the MYL4 ortholog in zebrafish causes extensive structural 16 Discussion disruption of sarcomere structure, particularly at Z-lines, where many macromolecular structures involved in cell From this case-control study, we first report that a novel signaling are located.18 Disruption of macromolecular SNP rs4968309 in MYL4 was associated with AF onset complexes might cause changes in regulatory processes and LAd in AF patients without structural heart disease. that lead to cell death and fibrosis. The extensive atrial Moreover, our study results extend the possibility of using remodeling caused by MYL4 disruption that we have MYL4 polymorphisms in risk prediction for AF recurrence already observed in our previous study15 likely contributes after cryoballoon ablation. to the loss of electrical and mechanical responses. Essential myosin light chains, MYL4 (also known by the In contrast to rare variants that typically have large abbreviations ALC-1 (for atrial light chain-1) and ELC a effects and can be identified in studies of familial AF, such (for essential light chain, atrial)), are essential myosin light as MYL4 E11K mutation15 and MYL4 (c.234delC) frame- chain that are expressed in cardiac and skeletal shift deletion,17 a previous study based on the large-scale muscles.28 The essential myosin light chains wrap around gene sequencing of Icelanders identified SNPs (rs185183057, the lever arm domain of myosin, and together with regula- rs28588212, rs117626672, rs117626672) in MYL4 that tory myosin light chains are necessary for the normal inter- predispose to AF.21,22 It suggested that common variants actions between myosin heavy chains and actin that are with low or modest effects and their association with AF responsible for cross-bridge movement and associated also should be identified. In the present study, 7 SNPs on contraction. The N-terminal domain is particularly involved MYL4 were screened and polymorphisms of this gene Advance Publication SNPs in MYL4 Associated With AF 7 could affect the development of AF. By comparing the and pathophysiology of atrial fibrillation: Relationships among allele and genotype frequencies of specific SNPs between clinical features, epidemiology, and mechanisms. Circ Res 2014; individuals with AF and controls, we first report that 114: 1453 – 1468. 2. Pellman J, Sheikh F. Atrial fibrillation: Mechanisms, therapeutics, patients who carry the CC genotype of rs4968309 and and future directions. Compr Physiol 2015; 5: 649. carry the A allele of rs1515751 tend towards AF onset and 3. Dobromir D, Niels V, Wehrens XHT. The ryanodine receptor there was an association between rs4968309 and LAd in channel as a molecular motif in atrial fibrillation: Pathophysio- the enrolled patients. This result indicated that these site logical and therapeutic implications. Cardiovasc Res 2011; 89: 734 – 743. polymorphisms may also affect the function of MYL4 and 4. Campuzano O, Brugada R. Genetics of familial atrial fibrillation. result in atrial remodeling and AF. Europace 2009; 11: 1267. Although pulmonary vein isolation is an established 5. Gutierrez A, Chung MK. Genomics of atrial fibrillation.Curr rhythm control therapy for AF, recurrence may occur in a Cardiol Rep 2016; 18: 1 – 13. 6. Hayashi K, Tada H, Yamagishi M. The genetics of atrial fibril- proportion of individuals with AF. Thus, it would be lation. Curr Opin Cardiol 2017; 32: 10 – 16. beneficial to predict which patients are more likely to benefit 7. Chelu MG, Satyam S, Subeena S, Sufen W, van Oort RJ, from the procedure. There are established risk factors of Skapura DG, et al. Calmodulin kinase II-mediated sarcoplasmic AF recurrence after catheter ablation such as hypertension, reticulum Ca2+ leak promotes atrial fibrillation in mice. J Clin Invest 2009; 119: 1940 – 1951. obesity, metabolic syndrome, dilation of the left , 8. Dawood D, Kannankeril PJ, Donahue BS, Gayle K, Tanya S, and persistent AF. Because of increasing data highlighting Haines JL, et al. Cardiac sodium channel (SCN5A) variants the important role of genetics in AF, genetic status has the associated with atrial fibrillation. Circulation 2008; 118: e668; potential to guide therapeutic strategies in the treatment of author reply e669. AF. In this study, both the univariable analysis and multiple 9. Dobrev D, Friedrich A, Voigt N, Jost N, Wettwer E, Christ T, et al. The G protein-gated potassium current I(K,ACh) is constitu- regression analysis with adjustment for traditional risk tively active in patients with chronic atrial fibrillation. Circulation factors demonstrated that patients with the CC genotype 2005; 112: 3697 – 3706. of rs4968309 had an increased susceptibility to AF recur- 10. Ellinor PT, Nam EG, Shea MA, Milan DJ, Ruskin JN, Macrae rence, which suggests that rs4968309 may help in preproce- CA. Cardiac sodium channel mutation in atrial fibrillation. Heart Rhythm 2008; 5: 99 – 105. dural consideration of efficacy and evaluation. Rs4968309 11. Reza W, Niels V, Stefan KB, Dobromir D, Stanley N. Recent is a non-coding SNP, and non-coding SNPs may play an advances in the molecular pathophysiology of atrial fibrillation. important role in disease development as enhancer elements, J Clin Invest 2011; 121: 2955. DNase hypersensitivity regions, and chromatin markers, 12. Lemoine MD, James Elber D, Patrice N, Denis C, Yan QX, 32 Philippe C, et al. Arrhythmogenic left atrial cellular electro- indirectly influencing the function of encode regions. physiology in a murine genetic long QT syndrome model. These SNPs are considered the signals for the causative Cardiovasc Res 2011; 92: 67 – 74. genes. The genes in closest proximity to these SNPs have, 13. Thibodeau IL, Xu J, Li Q, Liu G, Lam K, Veinot JP, et al. therefore, been investigated and significant information Paradigm of genetic mosaicism and lone atrial fibrillation: Physiological characterization of a connexin 43-deletion mutant has been obtained. However, the exact biological pathway identified from atrial tissue. Circulation 2010; 122: 236 – 244. between these non-coding SNPs and the emergence of AF 14. Hodgson-Zingman DM, Karst ML, Zingman LV, Heublein still remains unsolved. DM, Darbar D, Herron KJ, et al. Atrial natriuretic peptide frameshift mutation in familial atrial fibrillation. N Engl J Med Study Limitations 2008; 359: 158 – 165. 15. Peng W, Li M, Li H, Tang K, Zhuang J, Zhang J, et al. Dysfunc- First, some patients in this study were at high risk for AF tion of myosin light-chain 4 (MYL4) leads to heritable atrial because hypertension, diabetes, and obesity are the most cardiomyopathy with electrical, contractile, and structural commonly reported risk factors. Second, our findings were components: Evidence from genetically-engineered rats. J Am based on a relatively small number of participants, and Heart Assoc, doi:10.1161/JAHA.117.007030. 16. Orr N, Arnaout R, Gula LJ, Spears DA, Leong-Sit P, Li Q, et al. would be more credible in larger cohorts. The polymor- A mutation in the atrial-specific myosin light chain gene (MYL4) phisms of MYL4 may differ in different races. Third, the causes familial atrial fibrillation. Nat Commun 2016; 7: 11303. study only focused on observation of MYL4, which may 17. Gudbjartsson DF, Holm H, Sulem P, Masson G, Oddsson A, not be relevant to AF development with other genes. Magnusson OT, et al. A frameshift deletion in the sarcomere gene MYL4 causes early-onset familial atrial fibrillation. Eur Heart J 2017; 38: 27 – 34. Conclusions 18. Nattel S. Close connections between contraction and rhythm: A new genetic cause of atrial fibrillation/cardiomyopathy and what The C allele and CC genotype of rs4968309 on MYL4 were it can teach us. Eur Heart J 2017; 38: 35 – 37. 19. Sabater-Molina M, Perez-Sanchez I, Hernandez Del Rincon JP, associated with AF onset and recurrence. The A allele of Gimeno JR. Genetics of hypertrophic cardiomyopathy: A review rs1515751 was also associated with AF onset. The polymor- of current state. Clin Genet 2017; 93: 3 – 14. phism of MYL4 might be helpful in predicting AF onset 20. Olesen MS, Nielsen MW, Haunso S, Svendsen JH. Atrial fibril- and prognosis after ablation in AF patients without lation: The role of common and rare genetic variants. Eur J Hum Genet 2014; 22: 297 – 306. structural heart disease. 21. Gudbjartsson DF, Hannes H, Gudjonsson SA, Florian Z, Asmundur O, Arnaldur G, et al. Large-scale whole-genome Acknowledgment sequencing of the Icelandic population. Nat Genet 2015; 47: 435 – 444. This work was supported by the Chinese National Natural Science 22. Gudbjartsson DF, Holm H, Sulem P, Masson G, Oddsson A, Foundation grants no. 81670230, 81700291, and 81770226. Magnusson OT, et al. A frameshift deletion in the sarcomere gene MYL4 causes early-onset familial atrial fibrillation. Eur Conflicts of Interest Heart J 2016; 38: 27. 23. Calkins H, Hindricks G, Cappato R, Kim YH, Saad EB, None. Aguinaga L, et al. WITHDRAWN: 2017 HRS/EHRA/ECAS/ APHRS/SOLAECE Expert Consensus Statement on Catheter and Surgical Ablation of Atrial Fibrillation. J Arrhythmia 2017; References 50: 1 – 55. 1. Andrade J, Khairy P, Dobrev D, Nattel S. The clinical profile 24. Montalescot G, Sechtem U, Achenbach S, Andreotti F, Arden C, Advance Publication 8 ZHONG Y et al.

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