Influence of LGALS3 gene polymorphisms on susceptibility and prognosis of dilated cardiomyopathy in a Northern Han Chinese population

Accepted Manuscript Influence of LGALS3 gene polymorphisms on susceptibility and prognosis of dilated cardiomyopathy in a Northern Han Chinese population

Yuhui Zhang, Yunhong Wang, Mei Zhai, Tianyi Gan, Xuemei Zhao, Rongcheng Zhang, Tao An, Yan Huang, Qiong Zhou, Jian Zhang PII: DOI: Reference:

S0378-1119(17)30991-5 doi:10.1016/j.gene.2017.11.026 GENE 42334

To appear in:

Gene

Received date: Revised date: Accepted date:

9 July 2017 7 October 2017 8 November 2017

Please cite this article as: Yuhui Zhang, Yunhong Wang, Mei Zhai, Tianyi Gan, Xuemei Zhao, Rongcheng Zhang, Tao An, Yan Huang, Qiong Zhou, Jian Zhang , Influence of LGALS3 gene polymorphisms on susceptibility and prognosis of dilated cardiomyopathy in a Northern Han Chinese population. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Gene(2017), doi:10.1016/j.gene.2017.11.026

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ACCEPTED MANUSCRIPT Title: Influence of LGALS3 gene polymorphisms on susceptibility and prognosis of dilated cardiomyopathy in a Northern Han Chinese population Short title: LGALS3 polymorphisms associate with dilated cardiomyopathy

Abstract

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List of authors: Yuhui Zhang, MDa,*,#, Yunhong Wang, MDa,#, Mei Zhai, MMa, Tianyi Gan, MDa, Xuemei Zhao, MMa, Rongcheng Zhang, MDa, Tao An, MDa, Yan Huang, MMa, Qiong Zhou, MBa, Jian Zhang, MD, PhD a,*

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Background: Galectin-3 plays an important role in modulating cardiac inflammation and fibrosis.

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It also takes part in the pathways underlying cardiac remodeling. Therefore, LGALS3 gene, encoding galectin-3 protein, is a promising candidate for the genetic study of dilated

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cardiomyopathy (DCM). To date, there has been no research evaluating the association between

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LGALS3 gene polymorphisms and the susceptibility and prognosis of DCM.

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Methods and Results: Genotyping of 4 single nucleotide polymorphisms (SNPs) in the LGALS3

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gene, which were reported to be functional in the literature, was performed in 279 unrelated clinically diagnosed DCM patients and 363 apparently healthy controls from Northern Han

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Chinese population using iPLEX SNP Genotyping analysis on a Sequenom MassARRAY System. The frequency of G allelic polymorphism of rs1009977 and the C allelic polymorphism of rs4652 a

From the State Key Laboratory of Cardiovascular Disease, Heart Failure Center Fuwai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China. # The first 2 authors contributed equally to this work. *Corresponding authors: Jian Zhang, MD, PhD, 167 Beilishi Road, Beijing，100037, China. Tel: 0086-13911102015; Fax: 0086 (10) 8839 6180. E-mail: [email protected]. Yuhui Zhang, MD, 167 Beilishi Road, Beijing，100037, China. Tel: 0086-15901314243; Fax: 0086 (10) 8839 8170. E-mail: [email protected] Funding: The project was supported by the National Science Foundation for Distinguished Young Scholars of China (Grant No. 2012-GZ15).

ACCEPTED MANUSCRIPT were lower in DCM patients (OR=0.77, 95% CI [0.60-0.99], P=0.045; OR=0.79, 95%CI [0.63-0.99], P=0.042, respectively). The minor variants of rs1009977 and rs4652 were associated with low susceptibility of DCM under additive genetic models (P=0.045 and P=0.040,

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respectively). The AA genotype of both rs2274273 and rs4644 was associated with lower left

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ventricular ejection fraction (recessive model, P=0.018 for both; additive model, P=0.039 for

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both). The G variant of rs1009977 was related with lower serum galectin-3 level in DCM patients

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under three genetic models (additive model, P=0.020, dominant model, P=0.020, recessive model, P=0.037). The A variant of both rs2274273 and rs4644 was associated with lower level of

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galectin-3 in DCM patients under additive model (P=0.032 for both) and dominant model

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(P=0.012 for both). None of the 4 SNPs was associated with the cardiovascular or all-cause death

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rate of DCM. In Conclusion, LGALS3 gene polymorphisms might be associated with the susceptibility of DCM in a Northern Han Chinese population.

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Key Words: LGALS3; polymorphism; galectin-3; dilated cardiomyopathy.

ACCEPTED MANUSCRIPT Introduction The pathogenesis of Dilated cardiomyopathy (DCM) is still unfolding, with both genetic and non-genetic factors accounting for its development

[1]

. Inflammation and cardiac fibrosis are the

[2-5]

. Galectin-3, a beta galactoside binding lectin, plays an important role in

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etiologic origin

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key mechanisms involved in the development and progression of heart failure irrespective of its

[6,7]

. It contributes to the alteration of the myocardial

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proliferation and collagen synthesis

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modulating cardiac inflammation and fibrosis by promoting migration of macrophages, fibroblast

extracellular matrix and participates in the pathways underlying cardiac remodeling [8]. Therefore,

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LGALS3 gene, which encodes galectin-3 protein, is a promising candidate for the genetic study

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of DCM. To date, whether the genetic mutation of LGALS3 might be associated with the

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susceptibility and prognosis of DCM has been rarely reported. The basis of our hypothesis was that galectin-3 mediates the adverse cardiac remodeling

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through the pathological process of inflammation and fibrosis, therefore different functional

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variants of galectin-3 might affect genetic predisposition towards the development and prognosis

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of DCM. The 2 SNPs, rs4644 and rs4652, are non-synonymous, with rs4644 rendering residue 64 of galectin-3 changing from histidine to proline and rs4652 changing the threonine at residue 98 to a proline

[9]

. They lies in high LD with the SNP rs2274273 and all were reported to affect the

circulating galectin-3 protein level, [10] while rs1009977 was correlated with cognitive function at old age via inflammatory pathway

[11]

.This study was to investigate whether the four

polymorphisms in the LGALS3 gene were associated with the susceptibility and prognosis of DCM. We also measured serum galectin-3 level in the DCM patients to determine whether its

ACCEPTED MANUSCRIPT serum expression was correlated with the variants in the LGALS3 gene.

Methods Study Subjects

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Patients with DCM, who agreed to participate in the genetic study, were consecutively

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recruited from the heart failure center of Fuwai Hospital from March 2009 to April 2013. The

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inclusion criterion for enrollment was a clinical diagnosis of DCM with reduced ejection fraction [1]

. The exclusion criteria were

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(<50%) based on the definition of the ESC working group

non-dilated cardiomyopathy, coronary heart disease, primary valvular heart disease, congenital

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heart disease, pulmonary heart disease, pericarditis, tumor or immune system disorders. Clinical

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assessment, laboratory tests, echocardiography and coronary angiography (or coronary

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computerized tomographic angiography), as well as cardiac magnetic resonance imaging (CMRI) and cardiac radionuclide examination if clinically indicated, were used to make and rule out the

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diagnosis. All the patients’ electronic medical records included their familial history. Their

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relatives’ hospitalization information in our hospital were also recorded, so that we knew whether

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they were related or not.

Control subjects were selected randomly from a community-based echocardiographic survey study, which collected detailed demographic and medical information to investigate the prevalence and risk factors of cardiovascular disease in the Beijing area from March 2012 to August 2012. According to our inclusion criteria, these individuals were unrelated to one another and were apparently healthy as assessed by questionnaire, physical examination, serum biochemical testing and echocardiography. The exclusion criteria were history or symptoms of

ACCEPTED MANUSCRIPT cardiovascular disease, on any medications for cardiovascular disease, and diagnosis of diabetes mellitus. All subjects were of Northern Han Chinese origin and we ascertained the subjects’ ethnicity

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according to their resident identity cards. Echocardiography was performed by independent

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ultrasound staff who were blinded to the polymorphism analysis results. The echocardiography

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parameters, such as left ventricular ejection fraction and internal cardiac dimensions, were

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measured according to standard methods. The protocol was approved by the Ethics Committee of Fuwai Hospital, Chinese Academy of Medical Sciences and Peking Union of Medical College.

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All subjects provided written informed consent. The study complied with the Declaration of

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Helsinki.

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Single Nucleotide Polymorphisms (SNP) Genotyping Blood was taken into EDTA-containing receptacles. Genomic DNA was extracted from

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white blood cells in peripheral venous blood according to standard salting-out method. SNP

System

in

384-well

plates.

(Sequenom,

Inc.,

San

Diego,

CA;

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MassARRAY

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genotyping was performed by an iPLEX SNP Genotyping analyzer using the Sequenom

http://www.sequenom.com). All assays for the polymerase chain reaction (PCR) and associated extension reactions were designed by on-line Assay Design Suite software (Sequenom), and the primers were synthesized by Invitrogen (Thermo Fisher Scientific). PCR conditions, allele specific primer extension and analysis of their products were performed according to the standard method provided by the Sequenom MassARRAY SNP technology. The resulting mass spectra were processed and analyzed for peak identification and allele determination by the MassARRAY

ACCEPTED MANUSCRIPT Typer 4.0 software (Sequenom). To ensure that the obtained genotypes were valid, each of the 4 SNPs was genotyped in duplicate in 5% of the DNA samples. All results were in full agreement. Genotyping was performed blindly to all other data. Genotyping call rates were between 99.0%

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and 99.5%.

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Follow-ups

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The DCM patients were followed up either by visit, telephone or medical records in the 3rd

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month, the 6th month, the 1st year, and annually thereafter. The survival status and the etiology of death were recorded. The major parameters for prognostic evaluation are death from any-cause or

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death from cardiovascular diseases.

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Statistical Analysis

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Two-tailed independent t test was used to compare the difference of continuous variables between patients and control groups. Kruskal-Wallis H test was used to compare the difference of

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nonnormal continuous variables among genotypes under 3 genetic models. Comparison of

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categorical variables was performed by the chi-square test. Deviation from Hardy–Weinberg

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equilibrium (HWE) for each SNP in the control group was assessed by a chi-square goodness of fit test. The chi-square test was used to evaluate differences in the allelic and genotypic frequencies of cases and controls. Logistic-regression methods were used to analyze the effect of genotype specified by different genetic models or allele on the susceptibility of DCM. Kaplan–Meier plots and multivariate Cox regression analysis were used for survival analysis among subgroups defined according to genotype or allele. All the above analyses were performed using SPSS (Version 19.0; IBM). Linkage disequilibrium (LD) and haplotype blocks were

ACCEPTED MANUSCRIPT identified using Haploview4.2. The Haplo.stats program developed by R language version 3.2.2 (http://www.r-project.org) was used to perform haplotype analysis. The threshold of significance was set as p< 0.05. False discovery rate (FDR) control was used to adjust for multiple testing [12].

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Results

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A total of 642 unrelated participants comprising 279 patients (219 men and 60 women, mean

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age= 49.5y; SD±15.5y) and 363 controls (249 men and 114 women, mean age=63.3y; SD±7.7y)

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were recruited for this study. The clinical characteristics of the patients and controls are presented in Table 1.

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There was no departure from HWE for any of the SNPs in the control group (Supplemental

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Table 1). The G allele of rs1009977 and the C allele of rs4652 were less frequent in the DCM

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patients (Supplemental Table 2). Rs1009977 and rs4652 were significantly associated with low susceptibility of DCM in the additive genetic model (P=0.045 and P=0.040, respectively) and

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respectively) (Table 2).

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tended to reduce susceptibility of DCM in the recessive genetic model (P=0.052 and P=0.064,

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All the 4 SNPs were in strong LD and in the same LD block, with rs4644 lying in high linkage disequilibrium with rs2274273 (r2 = 0.974) (Figure 1). Four haplotypes with frequency > 1%

were

detected

in

haplotype

analysis.

The

T-C-A-G

haplotype

of

rs1009977-rs4644-rs4652-rs2274273 was the most prevalent. Compared with the T-C-A-G haplotype, haplotype G-A-C-A tended to reduce the risk of DCM but without significance (OR=0.75, 95%CI [0.56-1.01]，P=0.059) (Table 3 & Supplemental Table 3). Serum galectin-3 levels were measured in all the DCM patients (Table 4). The A variant of

ACCEPTED MANUSCRIPT both rs2274273 and rs4644 was associated with lower level of galectin-3 in DCM patients under additive model (P=0.032 for both) and dominant model (P=0.012 for both). The G variant of rs1009977 was consistently related with lower level of galectin-3 in DCM patients (additive

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model, P=0.020, dominant model, P=0.020, recessive model, P=0.037). The presence of the AA

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P=0.018 for both; additive model, P=0.039 for both) (Table5).

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genotype of both rs2274273 and rs4644 was associated with lower LVEF (recessive model,

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The average follow-up period was 20.6±14.6 months. A total of 60 patients died from any-cause and 57 died from cardiovascular diseases. Kaplan–Meier survival analysis and

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multivariate Cox regression analysis showed that no polymorphisms of the 4 SNPs or constructed

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haplotype in the LGALS3 gene had effect on overall death or cardiovascular death in DCM

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patients. (Table 6&7).

Discussion

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In this study, we had two major findings: 1) the G variant of rs1009977 and C variant of

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rs4652 might be associated with low susceptibility of DCM; 2) the AA genotype of both

patients.

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rs2274273 and rs4644 might be associated with lower left ventricular ejection fraction in DCM

DCM patients with G variant of rs1009977 have lower serum galectin-3 level, but it is not the case in the healthy subjects, indicating that this SNP might play a role by affecting the expression of LGALS3 gene only in the pathological state. The exact function of this SNP was still unknown. It is located in the near 5’gene region in the LGALS3 gene and whether it affects the protein expression and functionality in the disease state of DCM needs to be further explored.

ACCEPTED MANUSCRIPT The minor allele of SNP rs4652 is also associated with low susceptibility of DCM. It resides in one of the N-terminal repeated domains (YPSAPGAY, residue 94–101), suggesting that it may be involved in the regulation of galectin-3 secretion

[13,14]

. The carriage of C allele was reported [9]

.

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to be associated with a lower serum galectin-3 level in the rheumatoid arthritis patients

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However, in our study, we did not find a significant difference of serum galectin-3 levels among

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the genotypic groups of rs4652 in the DCM patients. We speculate that this is because serum

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galectin-3 level is related not only with genotype but also with the disease states and pathological processes, which are different between rheumatoid arthritis and DCM. Besides, though the

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variation of SNP rs4652 did not change the serum galectin-3 level, it might affect the disease

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susceptibility by changing the tissue level of galectin-3 in the myocardium, which needs to be

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proven in the future study.

The SNP rs2274273 lies in high LD with non-synonymous variant rs4644 (r2 = 0.974).

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Genetic variants of both rs4644 and rs2274273 were reported to change the serum level of [10]

. In our study the variants were also associated with

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galectin-3 protein in general population

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lower galectin-3 level in DCM patients. However, further analysis showed that the minor variant A allele of rs4644 and rs2274273 were associated with lower LVEF, conflicting with the change of serum level[15]. One explanation might be that though the variants lead to lower serum level of galectin-3, the tissue level of galectin-3 is the different case[6]. The SNP rs4644 codes for proline (allele C) or histidine (allele A) at residue 64 of the galectin-3. Amino acids Ala62-Tyr63 of galectin-3 harbor the cleavage site for matrix metalloproteinases (MMPs) and substitution of the subsequent amino acid histidine with proline at residue 64 resulted in resistance to cleavage by

ACCEPTED MANUSCRIPT MMPs

[16]

, which might result in increased level of galectin-3 in the myocardium. Therefore, it

would be helpful if the tissue expression of galectin-3 in the heart could be studied further in the future.

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Though the serum level of galectin-3 was an independent risk factor for prognosis of heart

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failure[17-19], survival analysis in our study demonstrated that no gene polymorphism of LGALS3

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was associated with prognosis of DCM. One explanation was that the gene mutation was not the

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only factor to determine the serum level of galectin-3. The level of galectin-3 was also determined by the severity of disease itself.

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This study has several limitations. Firstly, although we found the significant LGALS3 gene

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polymorphisms associated with the susceptibilty of DCM, we still could not exclude false

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positive results. Besides, the statistical significance in the genetic analysis is small, thus replication studies should be performed in another independent sample set in the future to

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confirm the findings. Secondly, this study did not assess the expression of LGALS3 gene in the

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tissue, therefore it could not be withdrawn from this study that the significant SNPs in the

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LGALS3 gene affected the gene expression in the heart tissue and were directly involved in the fibrosis and inflammation. Thirdly, we did not perform functional studies to analyze the effects of mutant alleles in LGALS3 gene. In a word, the exact mechanism underlying the association between LGALS3 gene polymorphisms and DCM could not be elucidated from the present study. To sum up, our study provided some clues regarding the potential clinical association of the LGALS3 gene with DCM. It is reasonable to initiate a more comprehensive survey to confirm the finding of this study. In addition, functional experiments to explore the underlying cellular

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biological mechanism of these SNPs need to be performed in the future.

ACCEPTED MANUSCRIPT Acknowledgement & Sources of Funding: This work was supported by the National Science Foundation for Distinguished Young Scholars of China (Grant No. 2012-GZ15).

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Disclosure:

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The authors have no conflicts of interest to disclose.

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Human subjects/informed consent statement:

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All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (Ethics Committee of Fuwai Hospital, Chinese Academy

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of Medical Sciences and Peking Union of Medical College) and with the Helsinki Declaration of

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1975, as revised in 2000. Informed consent was obtained from all patients for being included in

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the study.

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CLINICAL

AND

FIBROSIS-ASSOCIATED

PROGNOSTIC BIOMARKER.

VALUE

OF

RELATION

GALECTIN-3, WITH

A

CLINICAL

NOVEL AND

ACCEPTED MANUSCRIPT BIOCHEMICAL CORRELATES OF HEART FAILURE. J Am Coll Cardiol, 55(10, Supplement),

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A26.E243, doi:http://dx.doi.org/10.1016/S0735-1097(10)60244-6.

ACCEPTED MANUSCRIPT Figure Legend Figure 1 Block structure of LD for genotyped SNPs in the LGALS3 gene (Stronger correlations between these

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SNPs are noted by red color in the intersecting squares linking each pair of SNPs)

ACCEPTED MANUSCRIPT Table 1. Characteristics of participants Variable

DCM (n=279)

Control (n=363)

P Value

Age(years)

49.5±15.5

63.3±7.7

<0.001

Male, n (%)

219(78.5%)

249(68.6%)

0.005

Hypertension

76(27.2%)

175(48.2%)

<0.001

Smoking

137(49.1%)

205(56.5%)

0.064

Drinking

119(42.7%)

157(43.3%)

0.879

Body mass index (kg/m2)

24.1±4.4

25.9±3.5

<0.001

Systolic blood pressure (mm Hg)

110.6±17.2

139.9±19.5

Diastolic blood pressure (mm Hg)

71.5±12.3

82.4±11.4

LAD (mm)

46.4±8.0

33.2±4.2

LVEDD (mm)

70.2±10.0

46.3±4.6

LVEF (%)

29.4±7.5

68.7±6.6

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History, n (%)

95.9±37.1 484.2±161.3

NT-proBNP (pg/ml)

2860.2±2844.3

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NYHA functional class, n (%)

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<0.001 <0.001 <0.001

<0.001

63.9±142.2

<0.001

75.8±17.1

317.9±85.6

<0.001

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Uric acid (μmol/L)

<0.001

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Laboratory parameter Creatine (μmol/L)

<0.001

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Echocardiography parameter

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Physical examination

45(16.1%)

-

-

III

120(43.0%)

-

-

IV

114(40.9%)

-

-

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II

DCM, dilated cardiomyopathy; LAD, left atrial diameter; LVEDD, left ventricular end diastolic diameter; LVEF, left ventricular ejection fraction; NT-proBNP, N-terminal pro-B natriuretic peptide; NYHA, New York Heart Association.

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Values are presented as mean±SD.

ACCEPTED MANUSCRIPT Table 2. Distribution of LGALS3 genotypes in Northern Han Chinese DCM patients and control subjects SNP

rs1009977

Model

Contrast

DCM

Control

OR (95% CI)

P

Adjusted* OR

P

Value

(95% CI)

Value*

Dominant

GG+GT/TT

123/152

183/178

0.79(0.58-1.08)

0.136

0.78(0.58-1.07)

0.124

Recessive

GG/GT+TT

15/260

35/326

0.54(0.29-1.01)

0.052

0.54(0.29-1.02)

0.057

Additive

GG/GT/TT

15/108/152

35/148/178

0.77(0.60-0.99

0.045

0.77(0.60-0.99)

0.043

Dominant

AA+AG/GG

85/191

131/230

0.78(0.56-1.09)

Recessive

AA/AG+GG

7/269

16/345

0.56(0.23-1.38)

Additive

AA/AG/GG

7/78/191

16/115/230

Dominant

AA+AC/CC

85/191

Recessive

AA/AC+CC

Additive

0.147

0.209

0.57(0.23-1.41)

0.220

0.78(0.59-1.04)

0.096

0.78(0.59-1.05)

0.098

133/229

0.77(0.55-1.07)

0.117

0.76(0.55-1.07)

0.114

7/269

16/346

0.56(0.23-1.39)

0.212

0.57(0.23-1.41)

0.223

AA/AC/CC

7/78/191

16/117/229

0.77(0.58-1.03)

0.078

0.77(0.58-1.03)

0.078

Dominant

CC+AC/AA

173/103

248/114

0.77(0.56-1.07)

0.124

0.76(0.55-1.06)

0.111

Recessive

CC/AC+AA

36/240

67/295

0.66(0.43-1.03)

0.064

0.68(0.44-1.07)

0.093

Additive

CC/AC/AA

36/137/103

67/181/114

0.79(0.62-0.99)

0.040

0.79(0.63-0.99

0.045

CR

IP

0.78(0.56-1.09)

US

rs4652

0.147

AN

rs4644

T

5) rs2274273

5)

CI, confidence interval; DCM, dilated cardiomyopathy; OR, odds ratio; SNP, single nucleotide polymorphism.

AC

CE

PT

ED

M

* Adjusted for gender.

ACCEPTED MANUSCRIPT

Table 3. LGALS3 haplotype distribution and effect on DCM Haplotype

Total

DCM

Contr

(n=27

ol

9)

Score

P

Psim

Value

Score

P

*

Value

(n=36

Psim*

OR (95% CI)†

*

P

P

Value

Value

†

*†

3) TCAG

0.58

0.61

0.56

1.92

0.055

0.044

1.87

0.062

0.065

Base

-

-

GACA

0.18

0.16

0.20

-1.67

0.095

0.100

-1.66

0.097

0.086

0.75 (0.56-1.01)

TCCG

0.13

0.12

0.13

-0.39

0.699

0.698

-0.29

0.769

0.790

GCCG

0.09

0.08

0.10

-0.70

0.485

0.538

-0.71

0.475

0.491

score: haplo. score test; sim: simulation, number of simulation=1000; †: haplo.glm analysis.

AC

CE

PT

ED

M

AN

US

* Adjusted for gender.

0.288

0.86 (0.61-1.21)

0.387

0.437

T

0.061

0.289

IP

CI, confidence interval; DCM, dilated cardiomyopathy; OR, odds ratio.

CR

Haplotype constructed in the sequence: rs1009977-rs4644-.rs4652-rs2274273.

0.059

0.80 (0.53-1.21)

ACCEPTED MANUSCRIPT Table 4. Association of LGALS3 gene polymorphism with serum galectin-3 level in the DCM patients SNP

Model

Genotype DCM（N=279）

Dominant

Recessive

rs4644

Additive

Dominant

Recessive

rs4652

Additive

TT

23.1±13.8

GG+GT

20.3±9.6

TT

23.1±13.8

GG

17.4±6.6

GT+TT

22.1±11.2

AA

16.9±15.6

AG

20.3±8.3

GG

22.6±12.5

AA+AG

20.1±8.3

GG

22.6±12.5

AA

16.9±15.6

AG+GG

22.0±11.3

AA

16.9±15.6

AC

20.3±8.3

CC

22.6±12.5

AA+AC

20.1±8.3

CC

22.6±12.5

AA

16.9±15.6

AC+CC

22.0±11.3

CC

20.1±12.2

Recessive

IP

0.020

0.037

0.032

0.012

0.179

0.032

0.012

0.179

0.147

21.0±10.2

AA

23.3±13.7

CC+AC

20.9±10.2

AA

23.3±13.7

CC

20.1±12.2

AC+AA

22.1±11.4

AC

Dominant

CE

AC

T

20.8±9.4

CR

Additive

GT

US

rs2274273

0.020

AN

Recessive

17.4±6.6

M

Dominant

GG

ED

Additive

P Value

PT

rs1009977

Median±IQR

0.060

0.289

DCM, dilated cardiomyopathy; IQR, interquartile range; SNP, single nucleotide polymorphism.

ACCEPTED MANUSCRIPT Table 5. Association of LGALS3 gene polymorphisms with clinical characteristics of DCM patients SNP

Model

Genotyp

NT-proBNP (pg/ml)

LVEF(%)

LAD(mm)

LVEDD(mm)

e Median±IQR

P

Median±IQR

Value rs10099

Additive

GG

1222.0±3742.5

0.226

P

Median±IQR

Value 25.0±12.0

0.130

P

Median±IQR

Value 47.0±16.0

0.306

P Value

67.0±12.0

0.231

GT

29.5±10.0

45.0±9.8

28.0±10.8

46.0±9.0

Dominant

2171.6±2372.7

GG+GT

0.209

29.0±10.0

0.232

1964.0±3472.1 28.0±10.8 2171.6±2372.7 Recessive

GG

0.140

25.0±12.0

GT+TT

Additive

AA

3195.0±3801.3

0.762

73

0.039

28.5±10.0 1895.0±3763.0 2121.0±2505.0

AA+AG

0.618

28.0±10.0

28.0±10.0

1964.0±3677.0 GG

28.0±10.0

PT

GG

ED

AG

Dominant

25.0±7.0

M

rs22742

0.211

28.0±10.0 2128.8±2980.8

0.179

47.0±16.0

0.716

46.0±9.0

AN

1222.0±3742.5

45.0±10.0

US

TT

CR

TT

IP

2015.2±3538.4

T

77

0.744

70.0±13.0 68.0±12.0

67.0±12.0

0.731

69.0±13.0

0.133

77.0±21.0

45.0±9.0

66.0±12.3

46.0±9.0

70.0±12.0

45.0±9.0

0.088

70.0±13.0

45.0±10.0

55.0±16.0

68.0.±12.5

0.433

46.0±9.0

67.0±15.5

0.033

0.050

70.0±12.0

Recessive

AA

CE

2121.0±2505.0

0.683

25.0±7.0

0.018

55.0±16.0

0.109

77.0±21.0

0.226

3195.0±3801.3

AC

AG+GG

28.0±10.0

45.0±10.0

69.0±13.0

2044.0±3025.1

rs4644

Additive

AA

3195.0±3801.3

0.762

AC

25.0±7.0

0.039

55.0±16.0

0.133

77.0±21.0

28.5±10.0

45.0±9.0

66.0±12.3

28.0±10.0

46.0±9.0

70.0±12.0

0.033

1895.0±3763.0 CC Dominant

2121.0±2505.0

AA+AC

0.618

28.0±10.0

0.744

45.0±9.0

0.433

67.0±15.5

1964.0±3677.0 CC

28.0±10.0 2121.0±2505.0

46.0±9.0

70.0±12.0

0.050

ACCEPTED MANUSCRIPT Recessive

AA

0.683

25.0±7.0

0.018

55.0±16.0

0.109

77.0±21.0

0.226

3195.0±3801.3 AC+CC

28.0±10.0

45.0±10.0

69.0±13.0

2044.0±3025.1 Additive

CC

2264.4±3284.0

0.955

AC

26.5±9.0

0.467

46.0±10.8

0.934

71.0±12.8

29.0±9.0

46.0±10.0

69.0±14.0

29.0±10.0

45.0±10.0

69.0±12.0

0.664

AA Dominant

2021.6±2377.1

CC+AC

0.763

28.0±9.0

0.553

46.0±10.0

AA

29.0±10.0

45.0±10.0

CC

0.942

26.5±9.0

0.227

AC+AA

29.0±10.0 2067.1±3037.1

46.0±10.8

0.855

45.0±10.0

US

2264.4±3284.0

CR

2021.6±2377.1 Recessive

0.718

IP

2151.0±3283.1

T

2090.1±3248.9

69.0±14.0

0.603

69.0±12.0

71.0±12.8

0.388

69.0±12.0

AN

IQR, interquartile range; LAD, left atrial diameter; LVEDD, left ventricular end-diastolic diameter; LVEF, left ventricular ejection

CE

PT

ED

M

fraction; NT-proBNP, N-terminal pro-B-type natriuretic peptide; SNP, single nucleotide polymorphism.

AC

rs4652

ACCEPTED MANUSCRIPT Table 6. Effect of LGALS3 gene polymorphism on prognosis in Northern Han Chinese DCM patients SNP

Model

Contrast

Log-rank P

Univariate analysis HR (95% CI)

Multivariate analysis

P Value

Adjusted* HR (95%

P Value*

CI) overall death

rs4652

0.84(0.50-1.41)

0.512

0.94(0.55-1.60)

0.810

Recessive

GG/GT+TT

0.872

0.91(0.28-2.91)

0.872

1.30(0.39-4.27)

0.668

Dominant

AA+AG/GG

0.645

0.87(0.49-1.55)

0.645

Recessive

AA/AG+GG

0.642

1.39(0.34-5.72)-

0.644

Dominant

AA+AC/CC

0.645

0.87(0.49-1.55)

0.645

Recessive

AA/AC+CC

0.642

1.39(0.34-5.72)

0.644

Dominant

CC+AC/AA

0.378

1.28(0.74-2.21)

Recessive

CC/AC+AA

0.186

1.58(0.80-3.12)

Dominant

GG+GT/TT

0.335

0.77(0.45-1.31)

Recessive

GG/GT+TT

0.918

0.94(0.29-3.01)

Dominant

AA+AG/GG

0.558

Recessive

AA/AG+GG

Dominant

AA+AC/CC

Recessive

AA/AC+CC

0.610

Dominant

CC+AC/AA

0.489

Recessive

CC/AC+AA

0.92(0.52-1.64)

0.780

2.30(0.52-10.09)

0.270

0.92(0.52-1.64)

0.780

2.30(0.52-10.09)

0.270

0.379

1.45(0.83-2.55)

0.196

0.190

1.77(0.89-3.53)

0.106

0.337

0.84(0.49-1.46)

0.541

0.918

1.32(0.40-4.34)

0.651

0.84(0.47-1.51)

0.559

0.88(0.49-1.59)

0.666

0.610

1.44(0.35-5.91)-

0.612

2.28(0.52-10.00)

0.275

0.558

0.84(0.47-1.51)

0.559

0.88(0.49-1.59)

0.666

1.44(0.35-5.91)

0.612

2.28(0.52-10.00)

0.275

1.21(0.70-2.11)

0.490

1.36(0.77-2.40)

0.294

1.64(0.83-3.26)

0.154

1.83(0.91-3.66)

0.088

rs4652

M

rs4644

ED

rs2274273

AN

cardiovascular death rs1009977

T

0.511

0.150

IP

rs4644

GG+GT/TT

CR

rs2274273

Dominant

US

rs1009977

CI, confidence interval; DCM, dilated cardiomyopathy; HR, hazard ratio.

AC

CE

PT

* Adjusted for age, gender, history of hypertension, history of diabetes mellitus, smoking, drinking, and uric acid.

ACCEPTED MANUSCRIPT Table 7. Effect of LGALS3 haplotype on prognosis in Northern Han Chinese DCM patients Haplotype

Log-rank P

Univariate analysis HR (95% CI)

Multivariate analysis P

Adjusted* HR (95%

Value

CI)

P Value*

TCAG

0.186

0.63(0.32-1.25)

0.190

0.57 (0.28-1.13)

0.106

GACA

0.690

0.89(0.50-1.58)

0.690

0.94(0.53-1.67)

0.828

TCCG

0.333

1.30(0.77-2.20)

0.335

1.37(0.79-2.35)

GCCG

0.421

0.81(0.48 -1.37)

0.422

0.87(0.51 -1.48)

T

overall death

TCAG

0.150

0.61(0.31-1.21)

0.154

0.55(0.27-1.10)

0.088

GACA

0.600

0.85 (0.47-1.54)

0.600

0.89(0.49-1.62)

0.708

TCCG

0.451

1.23(0.72-2.09)

0.451

1.28(0.74-2.21)

0.383

GCCG

0.259

0.73(0.43-1.26)

0.262

0.78(0.45-1.35)

0.367

0.260

IP

0.597

cardiovascular

US

CR

death

AN

Haplotype constructed in the sequence: rs10932374-rs13003941-rs1595064- rs1595065-rs3748960. CI, confidence interval; DCM, dilated cardiomyopathy; HR, hazard ratio.

AC

CE

PT

ED

M

* Adjusted for age, gender, history of hypertension, history of diabetes mellitus, smoking, drinking, and uric acid.

M

AN

US

CR

IP

T

ACCEPTED MANUSCRIPT

AC

CE

PT

ED

Figure 1

ACCEPTED MANUSCRIPT

AC

CE

PT

ED

M

AN

US

CR

IP

T

Abbreviation: DCM, dilated cardiomyopathy; SNP ,single nucleotide polymorphisms; PCR, polymerase chain reaction; HWE, Hardy–Weinberg equilibrium; LD, linkage disequilibrium; FDR, false discovery rate; MMP, matrix metalloproteinases.

ACCEPTED MANUSCRIPT

AC

CE

PT

ED

M

AN

US

CR

IP

T

Highlights •A case-control study evaluated the association between LGALS3 gene polymorphisms and dilated cardiomyopathy in the Northern Han Chinese population. •Variants of 2 single nucleotide polymorphisms in the LGALS3 gene were associated with low susceptibility of dilated cardiomyopathy. • AA genotype of both rs2274273 and rs4644 might be associated with lower left ventricular ejection fraction in patients with dilated cardiomyopathy. • None of the 4 single nucleotide polymorphisms in the LGALS3 gene had effect on overall death or cardiovascular death in patients with dilated cardiomyopathy.